Neutralizing monoclonal antibodies against the Nogo-66 receptor (NgR) and uses thereof

ABSTRACT

The subject invention relates to isolated proteins, particularly monoclonal antibodies, which bind to the Nogo-66 receptor. Specifically, these antibodies have the ability to inhibit the binding of the natural ligand of the Nogo-66 receptor and neutralize the Nogo-66 receptor. These antibodies or portions thereof of the invention are useful for detecting NgR and for inhibiting NgR activity, for example in a human suffering from a disorder in which NgR or Nogo-66 activity is detrimental.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This is a continuation of U.S. patent application Ser. No. 14/013,916,filed on Aug. 29, 2013, which is a continuation of U.S. patentapplication Ser. No. 12/880,333, filed on Sep. 13, 2010, which is adivisional of U.S. patent application Ser. No. 11/943,770, filed on Nov.21, 2007, now U.S. Pat. No. 7,906,120, which claims priority to U.S.Provisional Patent Application No. 60/860,256, filed on Nov. 21, 2006,the entire contents of all of which are fully incorporated herein byreference.

TECHNICAL FIELD

The present application describes Nogo-66 receptor binding proteins,particularly monoclonal antibodies, which have the ability to bind tothe Nogo-66 receptor and neutralize the function of the Nogo-66receptor. These antibodies may therefore have utility in the treatmentof several states including but not limited to mammalian brain trauma,spinal cord injury, stroke, neurodegenerative diseases, andschizophrenia.

BACKGROUND INFORMATION

Axonal regeneration after injury within the mammalian central nervoussystem (CNS) is almost always impossible; the outcome depends on thebalance between the intrinsic ability of the nerve fibers in the CNS tore-grow, and the inhibitory factors within the CNS, localized in themicroenvironment of the lesion site, which actively prevent there-growth, and thus the regeneration of the injured fiber tracts.

It has been established that CNS myelin, generated by oligodendrocytes,is the most relevant non-permissive factor for axonal growth in theearly phase of an injury, by causing growth cone collapse in vitro aswell as in vivo, which results in the direct inhibition of axonoutgrowth (for review see: Lee et al., 2003). Only recently majorinhibitory factors on CNS myelin have been identified: oligodendrocytemyelin glycoprotein (OMgp), Myelin associated glycoprotein (MAG) andNogo-A (Domeniconi et al., 2002; reviews: Woolf & Bloechinger, 2002;McGee & Strittmatter, 2003; Lee et al., 2003). The latter proteincontains a domain Nogo-66; GrandPré et al., 2000), which exerts a maininhibitory function. Interestingly, all three inhibitory proteins showhigh expression levels in the CNS and interact with the same neuronalglycosylphosphatidylinositol (GPI) moiety-anchored receptor, the Nogo-66receptor, or NgR (Fournier et al., 2001). The Nogo-66 receptor, NgR, isa 473 amino acid glycosylphosphatidylinositol-linked protein. Itconsists of an N-terminal signal sequence, followed by 8 leucine-richrepeat domains, a leucine-rich repeat C-terminal domain (togetherforming the so-called ectodomain) and the GPI-anchoring domain. Throughthe GPI-anchor, NgR is linked to the external neuronal plasmalemma.

NgR itself belongs to a family of three CNS-enriched GPI-anchoredproteins (named NgR, NgR2 and NgR3) with about 40% sequence identity butvery similar overall structural organization (Barton et al. 2003; Laurenet al. 2003; Pignot et al. 2003). Although NgR is the only member knownto interact with multiple myelin-associated inhibitory molecules, MAGhas recently been shown also to interact with NgR2 (Venkatesh et al.2005). The function of the NgR homologues is currently not known. NgRitself is not expressed during early development in rodents or chick,but shows high expression levels in adult animals; NgR is expressed inmost if not all of the CNS regions, including the spinal cord (Hunt etal., 2002a,b). Spinal cord expression has been shown in chick (Fournieret al., 2001), rat (Hunt et al., 2002a) and mouse (Wang et al., 2002b)at both the mRNA and protein level. Within adult CNS tissue, NgR proteinis expressed in all mature neurons, including their axonal processes.Ligand binding to NgR initiates an intracellular signaling cascade,which results in axon outgrowth inhibition and growth cone collapse. AsNgR does not contain a transmembrane domain, signaling requires aco-receptor, which transduces the NgR/ligand interaction signal into thecell. The initial step in NgR signaling is its interaction with theco-receptors p75 or TROY (Wong et al., 2002; Shao et al., 2005; Park etal., 2005). A second co-receptor has been identified, called Lingo-1.Only a ternary complex between NgR, P75 or TROY and Lingo-1 constitutesthe functional signaling complex (Mi et al., 2004; Park et al., 2005).The outcome of this signaling is a rearrangement of the actincytoskeleton. In the neuron this actin cytoskeletal change causes aninhibition of axon outgrowth and induction of growth cone collapse.

In vitro, dorsal root ganglion cells from NgR (−/−) mice loose Nogo66binding capacity and are less responsive to the inhibitory effects ofNogo66, Fc-MAG, OMgp or myelin in a growth cone collapse assay (Kim etal., 2004). NgR (−/−) mice demonstrated increased regeneration ofbrainstem tracts, including rubrospinal and raphespinal tracts, afterpartial or complete spinal cord injury. Even after a completeexperimental transection of the spinal cord, the NgR (−/−) mice showedincreased functional recovery in an open field test. Followinghemisection and complete transection of the spinal cord, recovery in NgR(−/−) mice was significantly better than in homozygous (+/+) andheterozygous littermates (Kim et al., 2004).

The present application describes the generation of neutralizingmonoclonal antibodies against the NgR, which selectively compete forNogo-66 binding and that are expected to ameliorate disorders in whichNgR activity may be detrimental. The neutralizing monoclonal antibodiesof the present invention are expected, for example, to promote neuronalregeneration in the injured CNS, specifically after acute spinal cordinjury, brain trauma or neurodegenerative diseases such as for example,Huntington's chorea, Parkinson's disease, Alzheimer's disease ormultiple sclerosis.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1a and 1b : Antibody mAb50 and mAb51 binding to human and rat NgR.

FIG. 2: Competition of AP-Nogo66 binding to NgR-Fc by mAb50 and mAb51.

FIG. 3a : Competition of Nogo66 binding to human and rat NgR expressedon HEK293f cells by mAB50 and mAB51.

FIG. 3b : Competition of Nogo66 binding to human NgR expressed onHEK293f cells by mAB50 and mAB51.

FIG. 4: Binding of mAB50 and mAB51 to NTera 2 cells.

FIG. 5: Quantification of neurite outgrowth from NTera 2 cellaggregates.

FIG. 6: Deletion mutants of the hNgR.

FIG. 7: Competition of MAG-Fc binding to NgR-Fc.

FIG. 8: Rat dorsal root ganglion neurons under permissive and inhibitoryconditions and neutralization of Nogo66-induced neurite outgrowthinhibition by mAb50.

FIG. 9: Neutralization of Nogo66-induced neurite outgrowth inhibition bymAB50 and mAb51 in rat DRG cells.

LIST OF SEQUENCES

SEQ ID NO. 1a: Human NgR protein

SEQ ID NO. 1b: Human NgR nucleotide

SEQ ID NO. 2a: Rat NgR protein

SEQ ID NO. 2b: rat NgR nucleotide

SEQ ID NO. 3: Antibody Clone 50 VH

SEQ ID NO. 4: Antibody Clone 50 VL

SEQ ID NO. 5: Antibody Clone 51 VH

SEQ ID NO. 6: Antibody Clone 51 VL

SEQ ID NO. 7a: AP-Nogo-66 protein

SEQ ID NO. 7b: AP-Nogo-66 nucleotide

DETAILED DESCRIPTION

The present application relates to isolated binding proteins thatinteract with the Nogo receptor (NgR), in particular neutralizingmonoclonal antibodies that bind to and neutralize human and rat NgR.These antibodies have the capacity to compete with Nogo-66 for bindingto NgR. Other aspects of the present application include methods ofmaking, pharmaceutical compositions using the same, and methods of usingsuch binding proteins.

Antibodies

The principal embodiment of the present application comprises isolatedproteins or polypeptides that specifically bind to at least one epitopeof a Nogo-66 receptor (NgR). The term “isolated protein” or “isolatedpolypeptide” is a protein or polypeptide that by virtue of its origin orsource of derivation is not associated with naturally associatedcomponents that accompany it in its native state; is substantially freeof other proteins from the same species; is expressed by a cell from adifferent species; or does not occur in nature. Thus, a polypeptide thatis chemically synthesized or synthesized in a cellular system differentfrom the cell from which it naturally originates will be “isolated” fromits naturally associated components. A protein may also be renderedsubstantially free of naturally associated components by isolation,using protein purification techniques well known in the art. The term“Polypeptide” as used herein, refers to any polymeric chain of aminoacids. The terms “peptide” and “protein” are used interchangeably withthe term polypeptide and also refer to a polymeric chain of amino acids.The term “polypeptide” encompasses native or artificial proteins,protein fragments and polypeptide analogs of a protein sequence. Apolypeptide may be monomeric or polymeric.

The isolated proteins or polypeptides that specifically bind to at leastone epitope of a Nogo-66 receptor (NgR) are capable of inhibitingbinding of a ligand to said NgR. The Nogo-66 receptor, NgR, is a 473amino acid glycosylphosphatidylinositol-linked protein. It consists ofan N-terminal signal sequence, followed by 8 leucine-rich repeatdomains, a leucine-rich repeat C-terminal domain (together forming theso-called ectodomain) and the GPI-anchoring domain. Through theGPI-anchor, NgR is linked to the external neuronal plasmalemma.Preferred proteins of the present invention are monoclonal neutralizingantibody or antigen-binding fragment thereof that bind to at least oneepitope of the human NgR. Nogo 66 is one of the several major inhibitoryfactors on CNS myelin that induces inhibition of axon outgrowth andpromotes collapse of cone growth.

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprised of four polypeptide chains, two heavy(H) chains and two light (L) chains interconnected by disulfide bonds.An antibody is said to be “capable of binding” a molecule if it iscapable of specifically reacting with the molecule to thereby bind themolecule to the antibody. The term “epitope” is meant to refer to thatportion of any molecule capable of being bound by an antibody, which canalso be recognized by that antibody. Epitopes or “antigenicdeterminants” usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and have specificthree-dimensional structural characteristics as well as specific chargecharacteristics.

The term “antigen-binding fragment” of an antibody (or simply “antigenfragment”), as used herein, refers to one or more portions of anantibody that retain(s) the ability to specifically bind the receptorand activate or modulate it, respectively. It has been shown that theantigen-binding function of an antibody can be performed by fragments ofa full-length antibody. Examples of binding fragments encompassed withinthe term “antigen-binding portion” of an antibody include (i) a Fabfragment, a monovalent fragment consisting of the VL, VH, CL and CH1domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) an Fdfragment consisting of the VH and CH1 domains; (iv) an Fv fragmentconsisting of the VL and VH domains of a single arm of an antibody, (v)a dAb fragment (Ward et al. (1989) Nature 341:544-546), which consistsof a VH domain; and (vi) an isolated CDR. Furthermore, although the twodomains of the Fv fragment, VL and VH, are coded for by separate genes,they can be joined, using recombinant methods, by a synthetic linkerthat enables them to be made as a single protein chain in which the VLand VH regions pair to form monovalent molecules known as single chainFv (scFv) antibodies. (See, e.g., Bird et al. (1988) Science242:423-426; Huston et al. (1988) Proceedings of the National Academy ofScience USA 85:5879-5883.) Such scFv antibodies are also intended to beencompassed within the term antigen-binding portion of an antibody.Other forms of single chain antibodies, such as diabodies are alsoencompassed within the term. Diabodies are bivalent, bispecificantibodies in which VH and VL domains are expressed on a singlepolypeptide chain, but using a linker that is too short to allow forpairing between the two domains on the same chain, thereby forcing thedomains to pair with complementary domains of another chain and creatingtwo antigen binding sites on the same receptor or across two receptormolecules. (See, e.g., Holliger et al. (1993) Proceedings of theNational Academy of Science USA 90:6444-6448; Poljak et al. (1994)Structure 2:1121-1123.)

A “monoclonal antibody” as used herein is intended to refer to apreparation of antibody molecules, which share a common heavy chain andcommon light chain amino acid sequence, in contrast with “polyclonal”antibody preparations that contain a mixture of different antibodies.Monoclonal antibodies can be generated by several novel technologieslike phage, bacteria, yeast or ribosomal display, as well as classicalmethods exemplified by hybridoma-derived antibodies (e.g., an antibodysecreted by a hybridoma prepared by hybridoma technology, such as thestandard Kohler and Milstein hybridoma methodology ((1975) Nature256:495-497). The antibodies of the present invention were generated bystandard immunization/hybridoma technique in mice using NgR proteingenerated in a mammalian cell line.

A “neutralizing monoclonal antibody” as used herein is intended to referto a preparation of antibody molecules, which upon binding to thespecific antigen are able to compete and inhibit the binding of thenatural ligand for said antigen. In the specific case of the presentapplication, the neutralizing antibodies of the present invention arecapable to compete with Nogo66 for binding to the NgR, and to preventNogo66 biological activity or function that would result from binding ofNogo66 to NgR.

Preferably, the monoclonal neutralizing antibody of the presentapplication is a human antibody. The term “human antibody” refers toantibodies having variable and constant regions corresponding to, orderived from, human germline immunoglobulin sequences (e.g., see Kabatet al. Sequences of Proteins of Immunological Interest, Fifth Edition,U.S. Department of Health and Human Services, NIH Publication No.91-3242, 1991). The human antibodies of the present application,however, may include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample, in the CDRs and, in particular, CDR3. As used herein, the term“CDR” refers to the complementarity determining region within antibodyvariable sequences. There are three CDRs in each of the variable regionsof the heavy chain and the light chain, which are designated CDR1, CDR2and CDR3, for each of the variable regions. In various embodiments, theantibody is a recombinant antibody or a monoclonal antibody. The mostpreferred neutralizing antibodies of the present application arereferred to herein as mAb50 and mAb51 (ATCC No. PTA-8383 and PTA-8384,respectively). mAb50 and mAb51 antibodies and functional antibodyfragments, mAb50- and mAb51-related antibodies and functional antibodyfragments, and other antibodies and functional antibody fragments withequivalent properties to mAb50 and mAb51, such as high affinity bindingto NgR with low dissociation kinetics and high neutralizing capacity,are intended as part of the present invention.

The binding affinity and dissociation rate of an anti-NgR antibody ofthe present application to an immunogenic NgR polypeptide or fragmentthereof, may be determined by any method known in the art. For example,the binding affinity can be measured by competitive ELISAs, c RIAs,BIAcore or KinExA technology. The dissociation rate also can be measuredby BIAcore or KinExA technology. The binding affinity and dissociationrate are measured by surface plasmon resonance using, e.g., a BIAcore.

One of the preferred antibodies of the present application has at least90% amino acid sequence identity with a sequence comprising a heavychain variable region (VH region) comprising the sequence of SEQ ID NO:3 and a light chain variable region (VL region) comprising the sequenceof SEQ ID NO: 4, the mAB50 antibody. Another preferred embodiment has atleast 90% amino acid sequence identity with a sequence comprising aheavy chain variable region (VH region) comprising the sequence of SEQID NO: 5 and a light chain variable region (VL region) comprising thesequence of SEQ ID NO: 6, the mAB51 antibody.

Preferably, the mAb50 and mAb51 antibodies bind human NgR with a EC₅₀ ofless than 1×10⁻⁹ M, more preferably the antibodies bind to NgR with aEC₅₀ below 1×10⁻¹⁰, and most preferably the antibodies bind to NgR withan EC₅₀ below 4×10⁻¹¹ M.

It is intended that the anti-NgR antibodies mAb50 and mAb51 bind tohuman NgR in various forms, including pro-NgR, mature NgR and truncatedNgR. The antibodies mAb50 and mAb51 do not specifically bind to otherNgR homologues, like NgR2 or NgR3, or other LRR-containing proteins.However, the antibodies mAb50 and mAb51 do exhibit cross reactivity toNgR from other species, in particular rodents and more specifically ratNgRs. MAb52 through 62 additionally are cross-reactive to mouse NgR. Forexample, the antibodies bind to NgR from rat (IC₅₀ of both antibodiesfor rat NgR is about 3×10⁻¹¹ M).

It is also intended that the isolated binding proteins that interactwith (NgR) of the present application may be a glycosylated bindingprotein wherein the antibody or antigen-binding portion thereofcomprises one or more carbohydrate residues. Nascent in vivo proteinproduction may undergo further processing, known as post-translationalmodification. In particular, sugar (glycosyl) residues may be addedenzymatically, a process known as glycosylation. The resulting proteinsbearing covalently linked oligosaccharide side chains are known asglycosylated proteins or glycoproteins. Protein glycosylation depends onthe amino acid sequence of the protein of interest, as well as the hostcell in which the protein is expressed. Different organisms may producedifferent glycosylation enzymes (eg., glycosyltransferases andglycosidases), and have different substrates (nucleotide sugars)available. Due to such factors, protein glycosylation pattern, andcomposition of glycosyl residues, may differ depending on the hostsystem in which the particular protein is expressed. Glycosyl residuesuseful in the invention may include, but are not limited to, glucose,galactose, mannose, fucose, n-acetylglucosamine and sialic acid.Preferably the glycosylated binding protein comprises glycosyl residuessuch that the glycosylation pattern is human.

The antibodies of the present application comprise a heavy chainconstant region, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgDconstant region. Furthermore, the antibody can comprise a light chainconstant region, either a kappa light chain constant region or a lambdalight chain constant region. Preferably, the antibody comprises a kappalight chain constant region. Alternatively, the antibody portion can be,for example, a Fab fragment or a single chain Fv fragment. Replacementsof amino acid residues in the Fc portion to alter antibody effector'sfunction are known in the art (Winter, et al. U.S. Pat. Nos. 5,648,260;5,624,821). The Fc portion of an antibody mediates several importanteffector's functions e.g. cytokine induction, ADCC, phagocytosis,complement dependent cytotoxicity (CDC) and half-life/clearance rate ofantibody and antigen-antibody complexes. In some cases these effector'sfunctions are desirable for therapeutic antibody but in other casesmight be unnecessary or even deleterious, depending on the therapeuticobjectives. Certain human IgG isotypes, particularly IgG1 and IgG3,mediate ADCC and CDC via binding to Fcγ Rs and complement C1q,respectively. Neonatal Fc receptors (FcRn) are the critical componentsdetermining the circulating half-life of antibodies. In still anotherembodiment at least one amino acid residue is replaced in the constantregion of the antibody, for example the Fc region of the antibody, suchthat effector's functions of the antibody are altered.

Generation of Recombinant Proteins

For immunization and for the ELISA assays, as well as for the neuriteoutgrowth assays (described in the “Examples” section), solublerecombinant human and rat NgR's were produced. Also, the ligand Nogo66,fused to a alkaline phosphatase tag (AP-Nogo66) was generated and servedas inhibitory factor for neurite outgrowth assays as well as a ligand inthe ELISA and FACS studies (also described in the “Examples” section).

Human and Rat NgR

The human NgR protein was based on accession number AAG53612. Theprotein DNA (for amino acids 27 to 450) was cloned into a pSec vector(Ambion), and the protein was generated by stable expression in CHO-K1cells. The expressed receptor consisted of 424 amino acids of thecomplete protein (the amino acids 27 to 450), coupled to a Myc and 6×Histag at the C-terminus according to SEQ ID No. 1: Human NgR protein andSEQ ID NO. 1b: Human NgR nucleotide. Human secNgR 27-450 aa_D6_CHO-K1cells were cultivated in eight 40-chamber cell factories with 5000 mlUltraCHO serum-free medium (Cambrex Bio Science) per cell factory untilconfluence (ca. 5 days). Then the 40 l supernatant was centrifuged andconcentrated up to 500 ml with Hemoflow F columns (Fresenius MedicalCare). The concentrate was frozen at −80° C. For the proteinpurification the concentrated protein supernatant was filled to 1000 mlwith 500 ml 20 mM NaH₂PO₄; 140 mM NaCl; pH 7.4 and concentrated again to300 ml. The concentration process was repeated once again and finally300 ml 20 mM NaH₂PO₄; 140 mM NaCl; pH 7.4 was added to the concentrate.50 ml Ni-NTA-Superflow Fa. (Qiagen catalog #30430), equilibrated with 20mM NaH₂PO₄; 300 mM NaCl; pH 8.0 was added to the 600 ml concentrate,stirred for 1 h at 6° C., let settle down at 6° C., supernatantdiscarded and Ni-NTA beads filled in a column. Column was washed at roomtemperature with 10 CV 20 mM NaH₂PO₄; 300 mM NaCl; pH 8.0 followed by a5-10 CV 20 mM NaH₂PO₄; 300 mM NaCl; 10 mM Imidazole; pH 8.0. The columnwas eluted with 20 mM NaH₂PO₄; 300 mM NaCl; 100 mM Imidazole; pH 8.0 andcollect the UV-280 nm active peak. The eluate was subsequently dialyzedover night at 6° C. against 5 L 25 mM Tris/HCl; pH 7.0 and the dialysateloaded at room temperature on a Q-Sepharose column (column size 1.6 cm×3cm; volume 6 ml; Amersham Biosciences catalog #17-0510-01). Buffer A was50 mM Tris/HCl; pH 7.0. Buffer B was 50 mM Tris/HCl; 1M NaCl; pH 7.0, ata flow rate of 2 ml/min. Gradient was 0% B hold 5 CV; 0-50% B in 12 CV;50-100% B in 2 CV; hold 100% B 5 CV. The fraction size was 2.5 ml.Subsequently, the fractions were analyzed on SDS-PAGE, the fractionspooled due to their size on SDS-page and purity (the highest NgR-Hispurity for the high glycosilated NgR-His; with the highest NgR-Hispurity for the low glycosylated NgR-His). The pooled fractions weredialyzed once more against 20 mM NaH₂PO₄; 140 mM NaCl; pH 7.4 in a 12-14k Da dialysing tube at 6° C., the fraction filtered through a 0.2 μmsterile filter and stored at 6° C. for further use. For long timestorage the receptor fractions were aliquoted and stored at −80° C. Therat NgR DNA (accession number AAM46772) was cloned into pcDNA3.1(Invitrogen), and expressed and produced in a transient expressionsystem in HEK293F cells. The protein contained amino acids 27 through450 coupled to a 6×His tag according to SEQ ID NO. 2a: Rat NgR proteinand SEQ ID NO. 2b: rat NgR nucleotide. The production of the rat proteinwas through standard transient expression for 48-72 hours in HEK293Fcells. Cellular supernatant was harvested and the purification of theprotein followed similar steps as described above for the human protein.In some experiments proteins from R&D Systems were used. These includedhuman recombinant NgR/Fc chimera, Cat. Number 1208-NG and RecombinantMouse Nogo Receptor/Fc Chimera, Cat. Number 1440-NG.

Cell Surface Expression of NgR

For NgR expressed on cell surface, the full-length receptor sequence(rat AAM46772 and human AAG53612, respectively) comprising the completeopen reading frame from amino acids 1 through 473 was cloned intopcDNA4. The plasmids were transfected into CHO-K1 or HEK293 cells understandard procedures. Briefly, cells were seeded in Petri dishes in MEMmedium, transfected with Fugene 6 (Roche) according to the manufacturer.Selection was carried out with 150 μg/ml Zeozin for 2-3 weeks andprotein expression verified by FACS (see example 4 below).

For transient expression in HEK293F cells, cells were transfected insuspension according to the manufacturer (Invitrogen; Free StyleSystem), harvested after 48 to 72 hours and used for FACS studies (seeExamples section).

Production of AP-Nogo66

AP-Nogo66 (SEQ ID NO. 7a and SEQ ID NO.7b) was produced under standardconditions. Briefly, Nogo66 was cloned into the pAPTag5 vector, HEK293cells were transfected with the construct and selected with RPMIGlutamax+10% FCS, 150 μg/ml Zeocin. For protein production HEK293 cellswere cultivated in six 10 chamber cell factories with 1200 ml RPMI(Invitrogen) plus 10% FCS per cell factory until confluence (ca. 3days). Then the supernatant was discarded and 1200 ml Pro293a-CDM(Cambrex Bio Science) were filled in each cell factory. The cells werecultivated for further 3 days. Afterwards the 7200 ml supernatant wascentrifuged and concentrated up to 350 ml with Hemoflow F columns(Fresenius Medical Care). After addition of 1 mM PefablocSC (ROCHE) theconcentrate was aliquoted and frozen at −80° C.

Production of Antibodies and Antibody Generating Cell Lines

Antibodies of the application can be generated by immunization of asuitable host (e.g., vertebrates, including humans, mice, rats, sheep,goats, pigs, cattle, horses, reptiles, fishes, amphibians, and in eggsof birds, reptiles and fish). Such antibodies may be polyclonal ormonoclonal. To generate the antibodies of the present application, thehost is immunized with an immunogenic NgR polypeptide or fragmentthereof of the invention. The term “immunization” refers herein to theprocess of presenting an antigen to an immune repertoire whether thatrepertoire exists in a natural genetically unaltered organism, or atransgenic organism, including those modified to display an artificialhuman immune repertoire. Similarly, an “immunogenic preparation” is aformulation of antigen that contains adjuvants or other additives thatwould enhance the immunogenicity of the antigen.

Immunization of animals may be done by any method known in the art. See,e.g., Harlow and Lane, Antibodies: A Laboratory Manual, New York: ColdSpring Harbor Press, 1990. Methods for immunizing non-human animals suchas mice, rats, sheep, goats, pigs, cattle and horses are well known inthe art. See, e.g., Harlow and Lane and U.S. Pat. No. 5,994,619. In apreferred embodiment, the NgR antigen is administered with an adjuvantto stimulate the immune response. Such adjuvants include complete orincomplete Freund's adjuvant, RIBI (muramyl dipeptides) or ISCOM(immunostimulating complexes). Such adjuvants may protect thepolypeptide from rapid dispersal by sequestering it in a local deposit,or they may contain substances that stimulate the host to secretefactors that are chemotactic for macrophages and other components of theimmune system. Preferably, if a polypeptide is being administered, theimmunization schedule will involve two or more administrations of thepolypeptide, spread out over several weeks.

It is contemplated that the animal host is immunized with NgR associatedwith the cell membrane of an intact or disrupted cell and antibodies ofthe present application are identified by binding to an immunogenic NgRpolypeptide of the invention.

After immunization of the animal host with an NgR antigen, antibodiesand/or antibody-producing cells may be obtained from the animal. Ananti-NgR antibody-containing serum is obtained from the animal bybleeding or sacrificing the animal. The serum may be used as it isobtained from the animal, an immunoglobulin fraction may be obtainedfrom the serum, or the anti-NgR antibodies may be purified from theserum. Serum or immunoglobulins obtained in this manner are polyclonal,thus having a heterogeneous array of properties.

Antibody Producing Cell Lines

The present application also describes antibody-producing immortalizedhybridomas that may be prepared from the immunized animal. Preferably,the immunized animal is a non-human animal that expresses humanimmunoglobulin genes and the splenic B cells are fused to a myelomaderived from the same species as the non-human animal.

After immunization, the animal is sacrificed and the splenic B cells arefused to immortalized myeloma cells as is well known in the art. See,e.g., Harlow and Lane, supra. Preferably, the myeloma cells do notsecrete immunoglobulin polypeptides (a non-secretory cell line). Afterfusion and antibiotic selection, the hybridomas are screened using NgR,or a portion thereof, or a cell expressing NgR. Preferably, the initialscreening is performed using an enzyme-linked immunoassay (ELISA) or aradioimmunoassay (RIA), preferably an ELISA (an example of ELISAscreening is provided in the Examples section).

Anti-NgR antibody-producing hybridomas are selected, cloned and furtherscreened for desirable characteristics, including robust hybridomagrowth, high antibody production and desirable antibody characteristics,as discussed further below. Hybridomas may be cultured and expanded invivo in syngeneic animals, in animals that lack an immune system, e.g.,nude mice, or in cell culture in vitro. Methods of selecting, cloningand expanding hybridomas are well known to those of ordinary skill inthe art. In a preferred embodiment, the hybridomas are mouse hybridomas,as described above. In another preferred embodiment, the hybridomas areproduced in a non-human, non-mouse species such as rats, sheep, pigs,goats, cattle or horses. In another embodiment, the hybridomas are humanhybridomas, in which a human non-secretory myeloma is fused with a humancell expressing an anti-NgR antibody.

The present application also describes recombinant antibodies that aregenerated from single, isolated lymphocytes using a procedure referredto in the art as the selected lymphocyte antibody method (SLAM), asdescribed in U.S. Pat. No. 5,627,052, PCT Publication WO 92/02551 andBabcock, J. S. et al. (1996) Proc. Natl. Acad. Sci. USA 93:7843-7848. Inthis method, single cells secreting antibodies of interest, e.g.,lymphocytes derived from any immunized animals, are screened using anantigen-specific hemolytic plaque assay, wherein the antigen NgR, or afragment thereof, is coupled to sheep red blood cells using a linker,such as biotin, and used to identify single cells that secreteantibodies with specificity for NgR. Following identification ofantibody-secreting cells of interest, heavy- and light-chain variableregion cDNAs are rescued from the cells by reverse transcriptase-PCR andthese variable regions can then be expressed, in the context ofappropriate immunoglobulin constant regions (e.g., human constantregions), in mammalian host cells, such as COS or CHO cells. The hostcells transfected with the amplified immunoglobulin sequences, derivedfrom in vivo selected lymphocytes, can then undergo further analysis andselection in vitro, for example by panning the transfected cells toisolate cells expressing antibodies to NgR.

Antibodies Generated In Vitro

In vitro methods also can be used to make the antibodies described inthe present application, wherein an antibody library is screened toidentify an antibody having the desired binding specificity. Methods forsuch screening of recombinant antibody libraries are well known in theart.

The recombinant antibody library may be from a subject immunized withNgR, or a portion of NgR. Alternatively, the recombinant antibodylibrary may be from a naïve subject, i.e., one who has not beenimmunized with NgR, such as a human antibody library from a humansubject who has not been immunized with human NgR. Antibodies of thepresent application are selected by screening the recombinant antibodylibrary with the peptide comprising human NgR (e.g., a peptidecorresponding to a portion of hNgR) to thereby select those antibodiesthat recognize NgR. Methods for conducting such screening and selectionare well known in the art, such as described in the references in thepreceding paragraph. To select antibodies of the present applicationhaving particular binding affinities for hNgR, such as those thatdissociate from human NgR with a particular k_(off) rate constant, theart-known method of surface plasmon resonance can be used to selectantibodies having the desired k_(off) rate constant. To selectantibodies of the present application having a particular neutralizingactivity for hNgR, such as those with a particular IC₅₀, standardmethods known in the art for assessing the inhibition of hNgR activitymay be used.

Characteristics of the Antibodies

The mAB50 and mAB51 of the present application, or antigen-bindingportion thereof, bind human NgR, and dissociates from human NgR with ak_(off) rate constant of about 0.1 s⁻¹ or less, preferably 1×10⁻² s⁻¹ orless, more preferably 1×10⁻³ s⁻¹ or less, even more preferably 1×10⁻⁴s⁻¹ or less, most preferably 1×10⁻⁵ s⁻¹ or less as determined by surfaceplasmon resonance. The term “surface plasmon resonance”, as used herein,refers to an optical phenomenon that allows for the analysis ofreal-time biospecific interactions by detection of alterations inprotein concentrations within a biosensor matrix, for example using theBIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway,N.J.). For further descriptions, see Jönsson, U., et al. (1993) Ann.Biol. Clin. 51:19-26; Jönsson, U., et al. (1991) Biotechniques11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; andJohnnson, B., et al. (1991) Anal. Biochem. 198:268-277.

TABLE 1 Individual Biacore Kinetic Rate Parameters (Antigen: hNgR-Fcchimera) Captured On-rate Off-rate Kd antibody Isotype (M − 1s − 1) (s− 1) (M) mAb50 IgG2a, k 3.51 × 10⁵ 4.97 × 10⁻⁵ 1.41 × 10⁻¹⁰ mAb51 IgG2a,k 5.44 × 10⁵ 5.88 × 10⁻⁵ 1.08 × 10⁻¹⁰

The term “K_(on)”, as used herein, is intended to refer to the on rateconstant for association of an antibody to the antigen to form theantibody/antigen complex as is known in the art.

The term “K_(off)”, as used herein, is intended to refer to the off rateconstant for dissociation of an antibody from the antibody/antigencomplex as is known in the art. The term “K_(d)”, as used herein, isintended to refer to the dissociation constant of a particularantibody-antigen interaction as is known in the art.

Alternatively, the antibody mAb50 and mAb51 of the present application,or an antigen-binding portion thereof, may inhibit human NgR activitywith an IC₅₀ of about 1×10⁻⁶ M or less, preferably 1×10⁻⁷ M or less,preferably 1×10⁻⁸ M or less, more preferably 1×10⁻⁹ M or less, morepreferably 1×10⁻¹⁰ M or less, and most preferably 1×10⁻¹¹ M or less. Theterm IC₅₀, as used herein, is intended to refer to the concentration ofan antibody that competes for binding of the ligand Nogo66 to NgR.

Fusion Antibodies and Immunoadhesins

The present application also describes a fusion antibody orimmunoadhesin that may be made which comprises all or a portion of ananti-Nogo receptor-1 antibody of the present application linked toanother polypeptide. In some embodiments, only the variable region ofthe anti-Nogo receptor-1 antibody is linked to the polypeptide. In otherembodiments, the VH domain of an anti-Nogo receptor-1 antibody of thisapplication is linked to a first polypeptide, while the VL domain of theantibody is linked to a second polypeptide that associates with thefirst polypeptide in a manner that permits the VH and VL domains tointeract with one another to form an antibody binding site. In otherembodiments, the VH domain is separated from the VL domain by a linkerthat permits the VH and VL domains to interact with one another (seebelow under Single Chain Antibodies). The VH-linker-VL antibody is thenlinked to a polypeptide of interest. The fusion antibody is useful todirecting a polypeptide to a cell or tissue that expresses a Nogoreceptor-1 ligand. The polypeptide of interest may be a therapeuticagent, such as a toxin, or may be a diagnostic agent, such as an enzyme;that may be easily visualized, such as horseradish peroxidase. Inaddition, fusion antibodies can be created in which two (or more)single-chain antibodies are linked to one another. This is useful if onewants to create a divalent or polyvalent antibody on a singlepolypeptide chain, or if one wants to create a bispecific antibody.

One embodiment provides a labeled binding protein wherein an antibody orantibody portion of the present application is derivatized or linked toanother functional molecule (e.g., another peptide or protein). Forexample, a labeled binding protein of the present application can bederived by functionally linking an antibody or antibody portion of thepresent application (by chemical coupling, genetic fusion, noncovalentassociation or otherwise) to one or more other molecular entities, suchas a nucleic acid, another antibody (e.g., a bispecific antibody or adiabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent,and/or a protein or peptide that can mediate association of the antibodyor antibody portion with another molecule (such as a streptavidin coreregion or a polyhistidine tag).

Useful detectable agents with which an antibody or antibody portion ofthe present application may be derivatized include fluorescentcompounds. Exemplary fluorescent detectable agents include fluorescein,fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin and thelike. An antibody may also be derivatized with detectable enzymes, suchas alkaline phosphatase, horseradish peroxidase, glucose oxidase and thelike. When an antibody is derivatized with a detectable enzyme, it isdetected by adding additional reagents that the enzyme uses to produce adetectable reaction product. For example, when the detectable agenthorseradish peroxidase is present, the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which isdetectable. An antibody may also be derivatized with a nucleic acid,biotin, and detected through indirect measurement of avidin orstreptavidin binding.

Another embodiment of the present application provides a crystallizedbinding protein. The term “crystallized” as used herein, refer to anantibody, or antigen binding portion thereof, that exists in the form ofa crystal. Crystals are one form of the solid state of matter, which isdistinct from other forms such as the amorphous solid state or theliquid crystalline state. Crystals are composed of regular, repeating,three-dimensional arrays of atoms, ions, molecules (e.g., proteins suchas antibodies), or molecular assemblies (e.g., antigen/antibodycomplexes). These three-dimensional arrays are arranged according tospecific mathematical relationships that are well understood in thefield. The fundamental unit, or building block, that is repeated in acrystal is called the asymmetric unit. Repetition of the asymmetric unitin an arrangement that conforms to a given, well-definedcrystallographic symmetry provides the “unit cell” of the crystal.Repetition of the unit cell by regular translations in all threedimensions provides the crystal. See Giege, R. and Ducruix, A. Barrett,Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2ndea., pp. 20 1-16, Oxford University Press, New York, N.Y., (1999).

Preferably the present application describes crystals of whole anti-NgRantibodies and fragments thereof as disclosed herein, and formulationsand compositions comprising such crystals. In one embodiment thecrystallized binding protein has a greater half-life in vivo than thesoluble counterpart of the binding protein. In another embodiment thebinding protein retains biological activity after crystallization.

Crystallized binding protein of the invention may be produced accordingmethods known in the art.

Single Chain Antibodies

The present application includes a single chain antibody (scFv) thatbinds an immunogenic NgR of the invention. To produce the scFv, VH- andV-encoding DNA is operatively linked to DNA encoding a flexible linker,e.g., encoding the amino acid sequence (G1Y4-Ser), such that the VH andVL sequences can be expressed as a contiguous single-chain protein, withthe VL and VH regions joined by the flexible linker (see e.g., Bird etal. (1988) Science 242:423-42 6; Huston et al. (1988) Proc. Natl. Acad.Sci. USA 85: 5879-5883; McCafferty et al., 30 Nature (1990) 34 8:552-554). The single chain antibody may be monovalent, if only a singleVH and VL are used, bivalent, if two VH and VL are used, or polyvalent,if more than two VH and VL are used.

Chimeric Antibodies

The present application further includes a bispecific antibody orantigen-binding fragment thereof in which one specificity is for animmunogenic Nogo receptor-1 polypeptide of the present application. Forexample, a chimeric antibody can be generated that specifically binds toan immunogenic NgR polypeptide of the invention through one bindingdomain and to a second molecule through a second binding domain. Theterm “chimeric antibody” refers to antibodies which comprise heavy andlight chain variable region sequences from one species and constantregion sequences from another species, such as antibodies having murineheavy and light chain variable regions linked to human constant regions.The chimeric antibody can be produced through recombinant molecularbiological techniques, or may be physically conjugated together. Inaddition, a single chain antibody containing more than one VH and VL maybe generated that binds specifically to an immunogenic polypeptide ofthe invention and to another molecule that is associated withattenuating myelin mediated growth cone collapse and inhibition ofneurite outgrowth and sprouting. Such bispecific antibodies can begenerated using techniques that are well known for example, Fanger etal. Immunol Methods 4: 72-81 (1994) and Wright and Harris, 20 (supra).In some embodiments, the chimeric antibodies are prepared using one ormore of the variable regions from an antibody of the invention. Inanother embodiment, the chimeric antibody is prepared using one or moreCDR regions from said antibody. The term “humanized antibody” refers toantibodies which comprise heavy and light chain variable regionsequences from a nonhuman species (e.g., a mouse) but in which at leasta portion of the VH and/or VL sequence has been altered to be more“human-like”, i.e., more similar to human germline variable sequences.One type of humanized antibody is a CDR-grafted antibody in which humanCDR sequences are introduced into nonhuman VH and VL sequences toreplace the corresponding nonhuman CDR sequences.

Humanized Antibodies

Humanized antibodies are antibody molecules from non-human species thatbinds the desired antigen having one or more complementarity determiningregions (CDRs) from the non-human species and framework regions from ahuman immunoglobulin molecule. Known human Ig sequences are known in theart. Such imported sequences can be used to reduce immunogenicity orreduce, enhance or modify binding, affinity, on-rate, off-rate, avidity,specificity, half-life, or any other suitable characteristic, as knownin the art. Framework residues in the human framework regions may besubstituted with the corresponding residue from the CDR donor antibodyto alter, preferably improve, antigen binding. These frameworksubstitutions are identified by methods well known in the art, e.g., bymodeling of the interactions of the CDR and framework residues toidentify framework residues important for antigen binding and sequencecomparison to identify unusual framework residues at particularpositions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmannet al., Nature 332:323 (1988), which are incorporated herein byreference in their entireties.) Three-dimensional immunoglobulin modelsare commonly available and are familiar to those skilled in the art.Computer programs are available which illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidateimmunoglobulin sequence, i.e., the analysis of residues that influencethe ability of the candidate immunoglobulin to bind its antigen. In thisway, FR residues can be selected and combined from the consensus andimport sequences so that the desired antibody characteristic, such asincreased affinity for the target antigen(s), is achieved. In general,the CDR residues are directly and most substantially involved ininfluencing antigen binding. Antibodies can be humanized using a varietyof techniques known in the art, such as but not limited to thosedescribed in Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285(1992); Presta et al., J. Immunol. 151:2623 (1993), Padlan, MolecularImmunology 28 (4/5):489-498 (1991); Studnicka et al., ProteinEngineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973(1994).

Derivatized and Labeled Antibodies

An antibody or an antigen-binding fragment of the present applicationcan be derivatized or linked to another molecule (e.g., another peptideor protein). In general, the antibody or antigen-binding fragment isderivatized such that binding to an immunogenic polypeptide of theinvention is not affected adversely by the derivatization or labeling.For example, an antibody or antibody portion of the present applicationcan be functionally linked (by chemical coupling, genetic fusion,noncovalent association or otherwise) to one or more other molecularentities, such as another antibody (e.g., a bispecific antibody or adiabody), a detection reagent, a cytotoxic agent, a pharmaceuticalagent, and/or a protein or peptide that can mediate association of theantibody or antigen-binding fragment with another molecule (such as astreptavidin core region or a polyhistidine tag). Still further, anantibody or antigen-binding portion thereof may be part of a largerimmunoadhesion molecule, formed by covalent or non-covalent associationof the antibody or antibody portion with one or more other or differentproteins or peptides. Examples of such immunoadhesion molecules includeuse of the streptavidin core region to make a tetrameric scFv molecule(Kipriyanov et al. (1995) Human Antibodies and Hybridomas 6:93-101) anduse of a cysteine residue, a marker peptide and a C-terminalpolyhistidine tag to make bivalent and biotinylated scFv molecules(Kipriyanov et al. (1994) Molecular Immunology 31:1047-1058). Antibodyportions, such as Fab and F(ab′)₂ fragments, can be prepared from wholeantibodies using conventional techniques, such as papain or pepsindigestion, respectively, of whole antibodies. Moreover, antibodies,antibody portions and immunoadhesion molecules can be obtained usingstandard recombinant DNA techniques.

A derivatized antibody may be produced by crosslinking two or moreantibodies (of the same type or of different types, e.g., to createbispecific antibodies). Suitable crosslinkers include those that areheterobifunctional, having two distinctly reactive groups separated byan appropriate spacer (e.g. m-maleimidobenzoyl-N-hydroxysuccinimideester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkersare available from Pierce Chemical Company, Rockford, Ill.

A derivatized antibody may also be a labeled antibody. For instance,detection agents with which an antibody or antibody portion of theinvention may be derivatized are fluorescent compounds, includingfluorescein, fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, lanthanidephosphors and the like. An antibody also may be labeled with enzymesthat are useful for detection, such as horseradish peroxidase,galactosidase, luciferase, alkaline phosphatase, glucoseoxidase and thelike. In embodiments that are labeled with a detectable enzyme, theantibody is detected by adding additional reagents that the enzyme usesto produce a detectable reaction product. For example, horseradishperoxidase with hydrogen peroxide and diaminobenzidine. An antibody alsomay be labeled with biotin, and detected through indirect measurement ofavidin or streptavidin binding. An antibody may also be labeled with apredetermined polypeptide epitope recognized by a secondary reporter(e.g., leucine zipper pair sequences, binding sites for secondaryantibodies, metal binding domains, epitope: tags). An anti-Nogoreceptor-1 antibody or an antigen fragment thereof also may be labeledwith a radio-labeled amino acid. The radiolabel may be used for bothdiagnostic and therapeutic purposes. The radio-labeled anti-Nogoreceptor-1 antibody may be used diagnostically, for example, fordetermining Nogo receptor-1 levels in a subject. Further, theradio-labeled anti-Nogo receptor-1 antibody may be used therapeuticallyfor treating spinal cord injury.

Examples of labels for polypeptides include, but are not limited to, thefollowing radioisotopes or radionucleotides ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In,¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, ¹⁵³Sm. An anti-Nogo receptor-1 antibody or anantigen fragment thereof may also be derivatized with a chemical groupsuch as polyethylene glycol (PEG), a methyl or ethyl group, or acarbohydrate group. These groups may be useful to improve the biologicalcharacteristics of the antibody, e.g., to increase serum half-life or toincrease tissue binding. Also, a label for polypeptides can include anucleic acid, for example DNA for detection by PCR, or enhancing geneexpression, or siRNA to suppress gene expression in NgR-bearing cells ortissues.

The class and subclass of anti-Nogo receptor-1 antibodies may bedetermined by any method known in the art. In general, the class andsubclass of an antibody may be determined using antibodies that arespecific for a particular class and subclass of antibody. Suchantibodies are available commercially. The class and subclass can bedetermined by ELISA, Western Blot as well as other techniques.Alternatively, the class and subclass may be determined by sequencingall or a portion of the constant domains of the heavy and/or lightchains of the antibodies, comparing their amino acid sequences to theknown amino acid sequences of various classes and subclasses ofimmunoglobulins, and determining the class and subclass of theantibodies.

Inhibition of NgR Activity by Anti-NgR Antibodies

The anti-Nogo receptor-1 antibodies of the present application, or anantigen-binding fragment thereof, inhibit the binding of a ligand toNgR. The IC₅₀ of such inhibition can be measured by any method known inthe art, e.g., by ELISA, RIA, or functional antagonism. The IC₅₀ mayvary between 0.01 and 100 nM. Preferably, the IC₅₀ is between 1 and 10nM. More preferably, the IC₅₀ of anti-Nogo receptor-1 antibodies of thepresent invention, or an antigen-binding fragment thereof, is between0.1 nM and 1 nM. Most preferably, the IC₅₀ is below 0.1 nM.

Dual Variable Domain Antibodies

Dual variable domain (DVD) binding proteins as used herein, are bindingproteins that comprise two or more antigen binding sites and aretetravalent or multivalent binding proteins. The term “multivalentbinding protein” is used in this specification to denote a bindingprotein comprising two or more antigen binding sites. The multivalentbinding protein is preferably engineered to have the three or moreantigen binding sites, and is generally not a naturally occurringantibody. The term “multispecific binding protein” refers to a bindingprotein capable of binding two or more related or unrelated targets.Such DVDs may be monospecific, i.e capable of binding one antigen ormultispecific, i.e. capable of binding two or more antigens. DVD bindingproteins comprising two heavy chain DVD polypeptides and two light chainDVD polypeptides are referred to a DVD Ig. Each half of a DVD Igcomprises a heavy chain DVD polypeptide, and a light chain DVDpolypeptide, and two antigen binding sites. Each binding site comprisesa heavy chain variable domain and a light chain variable domain with atotal of 6 CDRs involved in antigen binding per antigen binding site.DVD binding proteins and methods of making DVD binding proteins aredisclosed in U.S. patent application Ser. No. 11/507,050 andincorporated herein by reference. It is intended that the presentinvention comprises a DVD binding protein comprising binding proteinscapable of binding NgR. Preferably the DVD binding protein is capable ofbinding NgR and a second target. The second target is selected from thegroup consisting of repulsive guidance molecule (RGM), Nogo-A, MAG,OMgp, CSPG. Among CSPG one may choose from aggrecan, brevican, versican,neurocan, phosphacan or Te38. Therefore, these examples comprisemeylin-derived inhibitors, as well as known neuronal co-receptors ofNgR.

Dual-Specific Antibodies

The present application also describes “dual-specific antibody”technology. Dual-specific antibodies may serve as agonists, antagonists,or both in different combinations. The term “agonist”, as used herein,refers to a modulator that, when contacted with a molecule of interest,causes an increase in the magnitude of a certain activity or function ofthe molecule compared to the magnitude of the activity or functionobserved in the absence of the agonist. The term “antagonist” or“inhibitor”, as used herein, refer to a modulator that, when contactedwith a molecule of interest causes a decrease in the magnitude of acertain activity or function of the molecule compared to the magnitudeof the activity or function observed in the absence of the antagonist.Particular antagonists of interest include those that block or modulatethe biological activity of Nogo-66. Antagonists and inhibitors ofNogo-66 may include, but are not limited to any molecules, preferablymonoclonal antibodies that interact with the Nogo-66 receptor (NgR).

It should be noted that the interaction with NgR may result in bindingand neutralization of the receptor or other ligands/cell membranecomponents, and may be useful for additive or synergistic functioningagainst multiple diseases.

The present application also describes NgR antibodies combined with NgRco-receptors, like NgR and p75, NgR and TROY, NgR and LINGO-1. It alsocomprises antibodies cross-reacting between NgR and its ligand and NgRand myelin-derived inhibitory factors. These may comprise antibodiescross-reacting between NgR and repulsive guidance molecule (RGM), NGRand Nogo-A, NGR and MAG, NgR and OMpg, NgR and CSPG. Among CSPG one maychoose from aggrecan, brevican, versican, neurocan, phosphacan or Te38.Therefore, these examples comprise meylin-derived inhibitors, as well asknown neuronal co-receptors of NgR.

The present application also describes dual specific antibodies betweenNgR and growth factor receptors including, but not limited to, nervegrowth factor (NGF), brain-derived neurotropic factor (BDNF), epidermalgrowth factor (EGF), granulocyte-colony stimulating factor (G-CSF),granulocyte-macrophage colony stimulating factor (GM-CSF),neurotrophins, platelet-derived growth factor (PDGF), erythropoietin(EPO), thrombopoietin (TPO), myostatin (GDF-8), Growth Differentiationfactor-9 (GDF9) basic fibroblast growth factor (bFGF or FGF2),glial-derived neurotrophic factor (GDNF), ciliary neurotrophic factor(CNTF).

Uses of the Antibodies

Given their ability to bind to human NgR, the neutralizing antibodies ofthe present application, or portions thereof, can be used to detecthuman NgR (e.g., in a biological sample, such as serum or plasma), usinga conventional immunoassay, such as an enzyme linked immunosorbentassays (ELISA), a radioimmunoassay (RIA) or tissue immunohistochemistry.The present application provides a method for detecting human NgR in abiological sample comprising contacting a biological sample with anantibody, or antibody portion, of the invention and detecting either theantibody (or antibody portion) bound to human NgR or unbound antibody(or antibody portion), to thereby detect human NgR in the biologicalsample. The antibody is directly or indirectly labeled with a detectablesubstance to facilitate detection of the bound or unbound antibody.Suitable detectable substances include various enzymes, prostheticgroups, fluorescent materials, luminescent materials and radioactivematerials. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;examples of suitable prosthetic group complexes includestreptavidin/biotin and avidin/biotin; examples of suitable fluorescentmaterials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; and examples of suitable radioactive material include ³H, ¹⁴C,³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, ¹⁵³Sm.

The antibodies and antibody portions of the present applicationpreferably are capable of neutralizing human NgR activity both in vitroand in vivo. Accordingly, such antibodies and antibody portions of theinvention can be used to inhibit Nogo-66 binding to NgR or the resultingactivity. In another embodiment, the present application provides amethod for reducing Nogo-66 activity or NgR activity in a subject,advantageously from a subject suffering from a disease or disorder inwhich NgR resulting activity is detrimental. The present applicationprovides methods for reducing NgR activity in a subject suffering fromsuch a disease or disorder, which method comprises administering to thesubject an antibody or antibody portion of the present application suchthat NgR activity in the subject is reduced. Preferably, the NgR ishuman and the subject is a human subject. Alternatively, the subject canbe a mammal expressing an NgR to which an antibody of the invention iscapable of binding. Still further the subject can be a mammal into whichNgR has been introduced. An antibody of the present application can beadministered to a human subject for therapeutic purposes. Moreover, anantibody of the present application can be administered to a non-humanmammal expressing an NgR with which the antibody is capable of bindingfor veterinary purposes or as an animal model of human disease.Regarding the latter, such animal models may be useful for evaluatingthe therapeutic efficacy of antibodies of the invention (e.g., testingof dosages and time courses of administration).

As used herein, the term “a disorder in which NgR activity isdetrimental” is intended to include diseases and other disorders inwhich the presence of NgR or the resulting activity in a subjectsuffering from the disorder has been shown to be or is suspected ofbeing either responsible for the pathophysiology of the disorder or afactor that contributes to a worsening of the disorder. Accordingly, adisorder in which NgR activity is detrimental is a disorder in whichreduction of NgR activity is expected to alleviate the symptoms and/orprogression of the disorder. Non-limiting examples of disorders that canbe treated with the antibodies of the invention include those disordersdiscussed in the section below pertaining to pharmaceutical compositionsof the antibodies of the invention.

It is recognized that NgR plays an important role in the pathologyassociated with a variety of diseases involving neurological diseasesassociated with neurodegeneration or inhibition of neuroregenerativeprocesses, resulting in paralysis. This includes Amytropic LateralSclerosis, Brachial Plexus Injury, Brain Injury, including traumaticbrain injury, Cerebral Palsy, Friedrich's Ataxia, Guillain-BarréSyndrome, Leukodystrophies, Multiple Sclerosis, Post Polio, SpinaBifida, Spinal Cord Injury, Spinal Muscle Atrophy, Spinal Tumors,Stroke, Transverse Myelitits. Furthermore it is recognized that NgRplays a role in dementia, senile dementia, mild cognitive impairment,Alzheimer-related dementia, Huntington's chorea, tardive dyskinesia,hyperkinesias, mania, Morbus Parkinson, steel-Richard syndrome, Down'ssyndrome, myasthenia gravis.

NgR and its ligands may also be involved in the generation ordevelopment of inflammatory or autoimmune states involving knowninflammatory elements (Teng & Tang, 2005; Fontoura & Steinmann, 2006).These diseases include, but are not limited to rheumatoid arthritis,osteoarthritis, juvenile chronic arthritis, septic arthritis, Lymearthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathy,systemic lupus erythematosus, Crohn's disease, ulcerative colitis,inflammatory bowel disease, insulin dependent diabetes mellitus,thyroiditis, asthma, allergic diseases, psoriasis, dermatitisscleroderma, graft versus host disease, organ transplant rejection,acute or chronic immune disease associated with organ transplantation,sarcoidosis, atherosclerosis, disseminated intravascular coagulation,Kawasaki's disease, Grave's disease, nephrotic syndrome, chronic fatiguesyndrome, Wegener's granulomatosis, Henoch-Schoenlein purpurea,microscopic vasculitis of the kidneys, chronic active hepatitis,uveitis, septic shock, toxic shock syndrome, sepsis syndrome, cachexia,infectious diseases, parasitic diseases, acquired immunodeficiencysyndrome, acute transverse myelitis, Huntington's chorea, Parkinson'sdisease, Alzheimer's disease, stroke, primary biliary cirrhosis,hemolytic anemia, malignancies, heart failure, myocardial infarction,Addison's disease, sporadic, polyglandular deficiency type I andpolyglandular deficiency type II, Schmidt's syndrome, adult (acute)respiratory distress syndrome, alopecia, alopecia areata, seronegativearthopathy, arthropathy, Reiter's disease, psoriatic arthropathy,ulcerative colitic arthropathy, enteropathic synovitis, chlamydia,yersinia and salmonella associated arthropathy, spondyloarthopathy,atheromatous disease/arteriosclerosis, atopic allergy, autoimmunebullous disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid,linear IgA disease, autoimmune haemolytic anaemia, Coombs positivehaemolytic anaemia, acquired pernicious anaemia, juvenile perniciousanaemia, myalgic encephalitis/Royal Free Disease, chronic mucocutaneouscandidiasis, giant cell arteritis, primary sclerosing hepatitis,cryptogenic autoimmune hepatitis, Acquired Immunodeficiency DiseaseSyndrome, Acquired Immunodeficiency Related Diseases, Hepatitis B,Hepatitis C, common varied immunodeficiency (common variablehypogammaglobulinaemia), dilated cardiomyopathy, female infertility,ovarian failure, premature ovarian failure, fibrotic lung disease,cryptogenic fibrosing alveolitis, post-inflammatory interstitial lungdisease, interstitial pneumonitis, connective tissue disease associatedinterstitial lung disease, mixed connective tissue disease associatedlung disease, systemic sclerosis associated interstitial lung disease,rheumatoid arthritis associated interstitial lung disease, systemiclupus erythematosus associated lung disease,dermatomyositis/polymyositis associated lung disease, Sjogren's diseaseassociated lung disease, ankylosing spondylitis associated lung disease,vasculitic diffuse lung disease, haemosiderosis associated lung disease,drug-induced interstitial lung disease, fibrosis, radiation fibrosis,bronchiolitis obliterans, chronic eosinophilic pneumonia, lymphocyticinfiltrative lung disease, postinfectious interstitial lung disease,gouty arthritis, autoimmune hepatitis, type-1 autoimmune hepatitis(classical autoimmune or lupoid hepatitis), type-2 autoimmune hepatitis(anti-LKM antibody hepatitis), autoimmune mediated hypoglycaemia, type Binsulin resistance with acanthosis nigricans, hypoparathyroidism, acuteimmune disease associated with organ transplantation, chronic immunedisease associated with organ transplantation, osteoarthrosis, primarysclerosing cholangitis, psoriasis type 1, psoriasis type 2, idiopathicleucopaenia, autoimmune neutropaenia, renal disease NOS,glomerulonephritides, microscopic vasulitis of the kidneys, lymedisease, discoid lupus erythematosus, male infertility idiopathic orNOS, sperm autoimmunity, multiple sclerosis (all subtypes), sympatheticophthalmia, pulmonary hypertension secondary to connective tissuedisease, Goodpasture's syndrome, pulmonary manifestation ofpolyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis,Still's disease, systemic sclerosis, Sjogren's syndrome, Takayasu'sdisease/arteritis, autoimmune thrombocytopaenia, idiopathicthrombocytopaenia, autoimmune thyroid disease, hyperthyroidism, goitrousautoimmune hypothyroidism (Hashimoto's disease), atrophic autoimmunehypothyroidism, primary myxoedema, phacogenic uveitis, primaryvasculitis, vitiligo acute liver disease, chronic liver diseases,alcoholic cirrhosis, alcohol-induced liver injury, choleosatatis,idiosyncratic liver disease, Drug-Induced hepatitis, Non-alcoholicSteatohepatitis, allergy and asthma, group B streptococci (GBS)infection, mental disorders (e.g., depression and schizophrenia), Th2Type and Th1 Type mediated diseases, acute and chronic pain (differentforms of pain), and cancers such as lung, breast, stomach, bladder,colon, pancreas, ovarian, prostate and rectal cancer and hematopoieticmalignancies (leukemia and lymphoma). The human antibodies, and antibodyportions of the present application can be used to treat humanssuffering from autoimmune diseases, in particular those associated withinflammation, including, rheumatoid spondylitis, allergy, autoimmunediabetes, autoimmune uveitis.

It is also known that NgR interacts with other proteins including butnot limited to proteins relevant to cell adhesion, cell migration, celltracking, axon path finding, and extracellular matrix proteins. Apotential therapeutic combination comprised in the present applicationmay include antibodies against NgR and semaphorins (in particularSema-1a, 1b; Sema-2a; Sema3A, B, C, D, E, F; Sema4A, D; Sema5A; Sema6D;Sema7A; Sema VA), plexins (Plexin-A1-4, Plexin-B1-3, Plexin-C1,Plexin-D1, Tim-2), neuropilins (neuropilin-1 and neuropilin-2),cadherins (E-cadherins and N-cadherins), netrins (netrin-1), ephrins(EphA3, 4, 6, 7, 8; B2, B3) Eph receptors, Eph ligands, Ig CAMs,tenascin-C, CSPGs, tenascin, Sema 3A, fibronectin, laminin-1, collagen(e.g. collagen-IV), Robo, Abl, N-Cadherin, L1, NCAM.

NgR has been described also to interact with the extracellular amyloidprecursor protein fragments (Aβ). Thus, the present applicationcomprises a combination among antibodies against NgR and Aβ species (Aβ1-40, Aβ 1-42, Aβ oligomers, Aβ multimers, Aβ globulomer). This type ofcombination therapy may be interesting for the treatment of Alzheimer'sdisease. The combination may translate into a dual effect on neuriteoutgrowth/neuroprotection and alleviation of plaque load and cognitiveperformance in AD patients. Axonal and dendritic loss is a very earlyhallmark of Alzheimer's disease and a combinatorial treatment may bevery efficacious.

Also, as previously discussed, dual-specific antibodies between any oneof the partners described above may be of use. Such antibodypreparations as described above may be useful for the treatment ofAlzheimer's disease, Parkinson's disease, spinal cord injury, traumaticbrain injury, multiple sclerosis, peripheral nerve injury,schizophrenia, depression, anxiety, as well as any plasticity andneurite growth and neurotoxicity related disease cited above.

The antibodies of the present application may also be combined withpeptides allowing the trans-membrane transfer to include intracellulartarget proteins. Such peptide sequences may include, but are not limitedto, tat, antennapedia, poly-args, some anti-microbial peptides. Suchpeptides may allow transfer through membranes, including cellular plasmamembranes, but also epithelia and endothelial membranes, including theblood-brain-barrier, gut mucosa, meninges, and others.

Such peptides may also allow entry of cell signaling inhibitors into thecells, which may include antibodies or small molecules against NgRsignaling molecules, including ROCK, small GTPases, actin and myelinstabilizer.

An antibody, or antibody portion, of the present application also can beadministered with one or more additional small molecule therapeuticagents useful in the treatment of disorders in which NgR activity isinvolved as discussed in the foregoing paragraphs. It should beunderstood that the antibodies of the present application or antigenbinding portion thereof can be used alone or in combination with anadditional agent, e.g., a therapeutic agent, said additional agent beingselected by the skilled artisan for its intended purpose. For example,the additional agent can be a therapeutic agent art-recognized as beinguseful to treat the disease or condition being treated by the antibodyof the present invention. The additional agent also can be an agent thatimparts a beneficial attribute to the therapeutic composition e.g., anagent that affects the viscosity of the composition. Preferredcombinations may include, but are not limited to, antipsychotic agentssuch as, but not limited to Risperidone, Olanzapine, Quetiapine,Phenothiazines (Chlorpromazine, Fluphenazine, Levomepromazine,Pericyanine, Perphenazine, Prochlorperazine, Promazine, Thioridazine,Trifluoperazine), Butyrophenones (Benperidol, Haloperidol), Zotepine,Loxapine, Aripiprazole, Sertoline, Ziprasidone, small molecularinhibitors of Rho kinase activity (ROCK), including compounds likefasudil, dimethylfasudil or any other ROCK inhibitor, small receptorligands against GABA A receptors or metabotropic glutamate receptors(mGluRs), non-steroidal anti-inflammatory drugs (NSAIDS),anti-inflammatory corticosteroids such as methylprednisolone.

Pharmaceutical Compositions of the Invention

The antibodies and antibody portions of the present application can beincorporated into pharmaceutical compositions suitable foradministration to a subject. The pharmaceutical compositions of thepresent application may include a “therapeutically effective amount” ora “prophylactically effective amount” of an antibody or antibody portionof the invention. A “therapeutically effective amount” refers to anamount effective, at dosages and for periods of time necessary, toachieve the desired therapeutic result. A therapeutically effectiveamount of the antibody or antibody portion may be determined by a personskilled in the art and may vary according to factors such as the diseasestate, age, sex, and weight of the individual, and the ability of theantibody or antibody portion to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the antibody or antibody portion areoutweighed by the therapeutically beneficial effects. “prophylacticallyeffective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired prophylactic result.Typically, since a prophylactic dose is used in subjects prior to or atan earlier stage of disease, the prophylactically effective amount willbe less than the therapeutically effective amount.

Typically, the pharmaceutical composition comprises an antibody orantibody portion of the invention and a pharmaceutically acceptablecarrier. As used herein, “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, that are physiologically compatible. Examples of pharmaceuticallyacceptable carriers include one or more of water, saline, phosphatebuffered saline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. In many cases, it will be preferable to includeisotonic agents, for example, sugars, polyalcohols (such as mannitol,sorbitol), or sodium chloride in the composition. Pharmaceuticallyacceptable carriers may further comprise minor amounts of auxiliarysubstances such as wetting or emulsifying agents, preservatives orbuffers, which enhance the shelf life or effectiveness of the antibodyor antibody portion.

The compositions of the present application may be in a variety offorms. These include, for example, liquid, semi-solid and solid dosageforms, such as liquid solutions (e.g., injectable and infusiblesolutions), dispersions or suspensions, tablets, pills, powders,liposomes and suppositories. The preferred form depends on the intendedmode of administration and therapeutic application. Typical preferredcompositions are in the form of injectable or infusible solutions, suchas compositions similar to those used for passive immunization of humanswith other antibodies. The preferred mode of administration isparenteral (e.g., intravenous, subcutaneous, intraperitoneal,intramuscular). In a preferred embodiment, the antibody is administeredby intravenous infusion or injection. In another preferred embodiment,the antibody is administered by intramuscular or subcutaneous injection.Yet another preferred embodiment includes the application of theantibody intrathecally.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the active compound (i.e.,antibody or antibody portion) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile,lyophilized powders for the preparation of sterile injectable solutions,the preferred methods of preparation are vacuum drying and spray-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The proper fluidity of a solution can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

The antibodies and antibody portions of the present application can beadministered by a variety of methods known in the art, although for manytherapeutic applications, the preferred route/mode of administration issubcutaneous injection, intravenous injection or infusion. As will beappreciated by the skilled artisan, the route and/or mode ofadministration will vary depending upon the desired results. In certainembodiments, the active compound may be prepared with a carrier thatwill protect the compound against rapid release, such as a controlledrelease formulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Robinson, ed.,Sustained and Controlled Release Drug Delivery Systems, Marcel Dekker,Inc., New York, 1978.

In certain embodiments, an antibody or antibody portion of the presentapplication may be orally administered, for example, with an inertdiluent or an assimilable edible carrier. The compound (and otheringredients, if desired) may also be enclosed in a hard or soft shellgelatin capsule, compressed into tablets, or incorporated directly intothe subject's diet. For oral therapeutic administration, the compoundsmay be incorporated with excipients and used in the form of ingestibletablets, bucal tablets, troches, capsules, elixirs, suspensions, syrups,wafers, and the like. To administer a compound of the invention by otherthan parenteral administration, it may be necessary to coat the compoundwith, or co-administer the compound with, a material to prevent itsinactivation.

EXAMPLES

The present application will be further clarified by the followingexamples, which are only intended to illustrate the present applicationand not to limit its scope in any way.

Example 1. Production of Antibodies

A/J mice (The Jackson Laboratories, Bar Harbor Me.) were immunized withrecombinant human and rat NgR protein (SEQ ID NO:1a and SEQ ID NO:2a,respectively. Mice were immunized 4 times subcutaneously with 50 ugrecombinant human or rat NgR protein in Complete Freund's adjuvant forthe first injection and Immuneasy™ (Qiagen) for the last threeimmunizations. Four days prior to fusion, mice were injected with bug ofantigen intravenously. For the fusion, spleen cells from immunizedanimals were fused with SP2/0-Ag14 myeloma cells at a ratio of 5:1 usingthe standard techniques of Kohler and Milstein (Kohler G and Milstein C;Nature Vol. 256, pages 495-497 (1975). Seven to ten days post fusion,when macroscopic hybridoma colonies were observed; supernatants weretested by ELISA assays. Recombinant human or rat NgR protein(s) at 1ug/ml in PBS were coated on ELISA plates overnight at 4° C. and blockedfor one hour at room temperature. Diluted supernatants were incubatedand binding was detected with Goat anti-mouse IgFc-HRP conjugate.Hybridoma cells producing antibody positive in the ELISA were scaled upand subcloned by limiting dilution. The isotype of the antibody wasdetermined using the Zymed EIA isotyping kit.

Different ELISA formats have been established and are routinely used asa first screen to identify antibodies that bind to the human, rat ormouse NgR. Antibodies that reacted in the ELISA assays were then testedfor binding to HEK or CHO cells that stably expressed recombinant humanNgR or recombinant rat NgR, and not their untransfected or controlcells. For the ELISA format, the soluble receptors were produced (seeabove). For the FACS studies, full-length NgR proteins were expressed inrecombinant cell lines. Results for Mab 50 SEQ ID NO: 3 and SEQ ID NO:4and Mab51 SEQ ID NO:5 and SEQ ID NO:6 in ELISA binding and FACS assaysare shown in Table 2.

TABLE 2 Summary of binding properties of Mab 50 and Mab 51 ELISA ELISAFACS FACS FACS FACS binding Binding binding binding binding bindingMonoclonal HuNgR 6X Rat NgR HEK293 HEK293 CHO K1 CHO K1 Antibody His 6xHis NGR Control Rat NGR Control Isotype Mab 50 Positive PositivePositive Negative Positive Negative IgG2a,k Mab 51 Positive PositivePositive Negative Positive Negative IgG2a,k

Additional antibodies Mab 1, Mab 52, Mab 53, Mab 54, Mab 55, Mab 56, Mab57, Mab 58, Mab 59, Mab 60, Mab 61, and Mab 62 were obtained using thesame experimental protocol.

Example 2. Determining Antibody Specificity and Binding Affinity byELISA

To determine the specificity of the monoclonal antibodies produced inExample 1, an ELISA using NgR-Fc (a fusion protein comprising aminoacids Met 1 to Ser 447 of the human NgR and a human Fc fragment (R&DSystems)) and rat NgR (SEQ ID NO. 2a), was performed. Both ligands wereimmobilized on 96-well microtiter plates (Nunc Maxisorb; 0.2 μg/well).As a blocking reagent 2% bovine serum albumin (BSA) in Tris-HCL, pH 7.2was used for 2 h at room temperature. The monoclonal antibodies wereused in concentrations starting at 10,000 ng/ml. Bound antibodies weredetected with a secondary anti-mouse antibody labeled with horseradishperoxidase (Sigma) and developed using 3,3′,5,5′-tetramethylbenzidinesubstrate (TMB, Pierce) under standard conditions. mAb50 and mAb51specifically bound to human NgR. When using 0.2 μg human NgR-Fc/well a50% binding was seen for both monoclonal antibodies at concentrations ofless then 20 ng/ml (FIG. 1A). In similar experiments, mAb50 and mAb51also specifically bound to a polypeptide consisting of amino acids ofthe rat NgR. When using 0.2 μg rat NgR; well of a 96-well microtiterplate a 50% binding was seen for both monoclonal antibodies atconcentrations of less then 20 ng/ml (FIG. 1B) The rest of themonoclonal antibodies (mABs) of the present invention also bound to thehuman and to the rat NgR. Differences in the signals obtained with eachantibody suggested differences in binding affinities (See Table 3)

TABLE 3 Summary of EC50 values for mAbs using human or rat NgR EC50human NgR rat NgR mAb ng/ml ng/ml 1 10 10 4 8 >100 52 3 4 53 3 2 54 5 455 2 10 56 8 20 57 20 >100 58 5 15 59 15 15 60 2 5 62 10 7

Example 3. Characterization of Antibody Binding to Soluble Human and RatNgR Using Dot Blots and Western Blots

For dot blots, 2 al protein in different concentration were spotted inTTBS buffer onto dry nitrocellulose membrane. For Western blotsfilterpaper and nitrocellulose were soaked for 10 min in Novex transferbuffer with 20% methanol. Blotting was achieved in a Novex chamber atconstant current (100 mA) for 2 hrs at room temperature.

The amount of protein added per spot was:

a) 100 μg/ml≈200 ng/spot

b) 50 μg/ml≈100 ng/spot

c) 10 μg/ml≈20 ng/spot

d) 5 μg/ml≈10 ng/spot

e) 1 μg/ml≈2 ng/spot

f) 500 ng/ml≈1 ng/spot

After spotting the probes, the membrane dried for 10 min at roomtemperature before starting the immunodetection protocol.

All tested monoclonal antibodies bound to the human and to the rat NgR.Differences between signals obtained with different antibodies suggesteddifferences in binding affinity. (MAB61 did show high background onblots under our conditions used for probing the nitrocellulosemembranes.) Binding was antibody dependent since omitting the antibodiesdirected against the NgR “control” did not show any signal.

The monoclonal antibodies reacted differently to the denatured NgR onWestern Blots. Only MAB1 did show prominent signals to the human as wellas to the rat NgR. As a control for the binding ability of theantibodies dots containing non-denatured human-NgR and non-denaturedrat-NgR were spotted onto the nitrocellulose membrane after the proteinswere transferred from the SDS gels. These dots served as positivecontrols confirming binding of all monoclonal antibodies to thenon-denatured proteins on the same nitrocellulose membrane. Results aresummarized in Table 4.

TABLE 4 Dot blot and Western blot analyses. Dot Blot Western Blotantibody hNgR rNgR hNgR rNgR MAB1 +++ ++ +++ ++ MAB52 +++ +++ − − MAB53+++ ++ ~ − MAB54 ++ + − − MAB55 +++ ++ ~ − MAB56 ++ + − − MAB57 ++ ~ − −MAB58 ++ + − − MAB59 +++ ++ ~ − MAB60 +++ ++ ~ − MAB61 ++ + − − MAB62+++ ++ ~ − +++: very strong signal; ++: strong signal; +: signal; −: nosignal; ~: no relevant signal.

Example 4. Competition of AP-Nogo66 Binding to Soluble Human NogoReceptor (hNgR-Fc)

To further characterize the monoclonal antibodies a competition assaysimilar to the binding assay of Example 2 was used to test the abilityof the mAbs produced in Example 1 to inhibit AP-Nogo66 binding to humanNgR-Fc. Human NgR-Fc (0.2 μg/well) was immobilized on 96-well microtiterplates (Nunc Maxisorb) in 50 mM Na-carbonate buffer pH 9 over night at4° C. followed by a 2 h blocking step with 2% BSA in Tris-HCL, pH 7.2 atroom temperature. The wells received a constant concentration ofAP-Nogo66 (final concentration 0.15 nM in Tris-HCL, pH 7.2 with 0.1%BSA) and increasing concentrations of the mAbs. Plates were incubatedfor 90 min at room temperature. During each incubation step plates werewashed with washing buffer (10 mM Tris-HCl, pH7.2 and 0.05% Tween20).Binding of AP-Nogo66 was detected with the AttoPhos substrate (Roche)and the fluorescence units were measured in the Polarstar (BMG)instrument. FIG. 2 shows the relative fluorescence units (RFU) formeasurements taken at 0 min and 30 min for mAb 50 and mAb 51. Thebinding was completely blocked by mAb 50 and mAb 51, and a 10-fold molarexcess of both antibodies was required to inhibit 50% of AP-Nogo66binding to human NgR-Fc. The remaining antibodies, mab 52, mAb 53, mAb54, mAb 55, mAb 56, mAb 57, mAb 58, mAb 59, mAb 60, mAb 61, and mAb 62except mAb1, blocked the binding of AP-Nogo66 to soluble Nogo receptor,in a similar concentration range as seen for mAb 50 and 51.

Example 5. Competition of AP-Nogo66 Binding to Human and Rat NgR onHEK293f Cells by mAB50 and mAb51

To further characterize the binding and competitive properties of mAbsproduced as described in Example 1, a suspension of HEK293f cellstransiently expressing human or rat NgR was used. 48 h aftertransfection the cells were plated in 96-well microtiter plates (NuncMaxisorb) and washed with Phosphate buffered saline buffer (PBScontaining 1% BSA). 1 μg/200 μl of AP-Nogo66 (final concentration 40 nM)and varying concentrations of monoclonal antibodies (20 μg, 4, 0.8 and0.16 μg/200 μl) were added and incubated for 1 h at 4° C. A monoclonalantibody with the same isotype was used as a negative control. Thebinding of AP-Nogo66 was detected with an anti-alkaline Phosphataseantibody (Sigma), which was labeled with Alexa Fluor using the Zenonmouse IgG2a labeling kit (Invitrogen). The secondary antibody wasincubated for 1 h at 4° C. At the end of the incubation, the cells werewashed with PBS and subjected to FACS analysis. At the 20 μg/200 μlconcentration (20 fold molar excess) polyclonal anti-NgR antibody andthe NgR-Fc (both from R&D Systems) blocked the AP-Nogo66 binding between60% and 70%, while mAb 50 and 51 blocked the AP-Nogo66 binding byapproximately 90%. The antibody isotype control is shown in FIG. 3a formAb 50 and mAB 51 in rat and human NgR at a concentration of 20 μg. Theantibody isotype control is shown as black lines (leftmost signal) andthe AP-Nogo66 binding in the absence of any antibody is shown as redlines (rightmost signal). Both human and rat NgR bound AP-Nogo66 equallywell. The AP-Nogo66 fluorescence is shifted to lower intensities(shaded; green area) in the presence of both antibodies in HEK293f cellsexpressing human and rat NgR. Using different antibody concentrations anIC₅₀ was seen with a 2-fold molar excess of monoclonal antibodies 50 and51 versus AP-Nogo66 for both the human and rat NgR as determined by theKolmogorov-Smirnov (K-S) assay.

FIG. 3b shows the results with varying concentrations of mAb 50 and mAb51, i.e. 20.0 μg, 4.0, 0.8 and 0.16 μg/200 μl. The other mAbs of theinvention blocked binding of AP-Nogo66 to Nogo receptor expressed onHEK293f cells, in a similar concentration range as seen for mAb 50 and51. See Table 5, which indicates the amount of antibody required for a50% competition of AP-Nogo66 binding human NgR expressed on HEK293fcells.

TABLE 5 Competition of AP-Nogo 66 to human and rat HEK293f cells. mAb50% Competition (μg) 50 0.5 51 0.5 52 3 53 3 54 0.8 55 0.6 56 0.8 57 0.258 0.2 59 3 60 0.8 62 0.3

Example 6. Neutralization of AP-Nogo66-Induced Neurite OutgrowthInhibition by mAB50 and mAb51 in NTera2 Cells

Antibodies of the present invention were studied for their effects onneurite outgrowth in a functional system closely resembling the in vivosituation using human NTera-2 cells and rodent (mouse cortical neurons,rat cortical neurons and rat cerebellar granule neurons) cell types, andAP-Nogo66 and myelin as ligands.

NTera2 cells (a human teratocarcinoma cell line) can be differentiatedinto neuron-like cells using retinoic acid, that express NgR mRNA (PCR)and cell surface protein as shown by modest binding of a polyclonalantibody to NgR (FACS binding). The expression of the natural human NgRby NTera-2 cells on the cell surface was detected by FACS analysis usingan isotype control antibody (unshaded area) and mAb 50 and mAb 51(shaded area), respectively (FIG. 4). The binding of both antibodies tothe NgR expressed on the surface of the Ntera-2 cells is shown by theshaded areas.

Analysis of neurite outgrowth or growth cone collapse in thesedifferentiated cells is an in vitro system as close as possible to thehuman neuron. NTera2 cells (from the German National Resource Center forBiologicals, DMSZ, Braunschweig) were thawed and plated in 175 cm²culture flasks (Greinerbio-one #660175) with DMEM medium (Gibco#31966-021+10% fetal calf serum (FCS)+5% Horse Serum (PS)). Afterseveral days in culture the cells were replated. For this the cells werewashed once with PBS (Gibco #14190-094), washed with trypsin/EDTA (Gibco#25300-054) and incubated with trypsin/EDTA for 5 minutes. Fordifferentiation, the cells were resuspended, 2.5×10⁶ cells were platedon 175 cm² flasks in DMEM (Gibco 31966-021, Lot. Nr. 3092594) plus 10%FCS+ 5% PS, plus 1% Penicillin/Streptomycin (5000/5000 units/mL), plusretinoic acid (SIGMA #R2625) at a final concentration of 10 μM).

For differentiation, retinoic acid (SIGMA #R2625) at a finalconcentration of 10 μM was added twice-weekly to the NTera2 cells over aperiod of three weeks. After 21 days differentiation the cells werereplated. For this the cells were washed once with PBS, washed withtrypsin/EDTA and incubated with trypsin/EDTA for 5 minutes. The cellswere resuspended, split 1:6 and plated on six 175 cm² flasks in DMEM(Gibco 31966-021, Lot. Nr. 3092594)+10% FCS+5% PS+1% PenStrep) for 2-3days.

After 2-3 days cells were washed with PBS, physically detached,centrifuged for 5 minutes at 1000 rpm, resuspended in Neurobasal medium(Gibco #21103-049) plus 2 mM L-Glutamine (Gibco #25030-024) plusPenicillin/Streptomycin plus B27-Supplement) and pre-aggregated in anErlenmeyer flask (Corning #431143). Thereby 10⁶ cells/mL were added to2×15 mL aggregation medium (Neurobsalmedium (Gibco #21103-049) plus 2 mML-Glutamine (Gibco #25030-024) plus Penicillin/Streptomycin plusB27-Supplement)), gently agitated over night at 37° C., 5% CO2 andplated on 96 well plates (Biocoat Poly-D-Lysin Cellware 96-WellBlack/Clear Plate Becton Dickinson #35 4640 (35 6640)) pre-coated withinhibitory and control substrates. For the inhibitory substrates half ofthe 96 well plate was pre-coated with 100 μL AP-Nogo66 (concentrationAP-Nogo66 was 15 μg/mL) plus Laminin (Sigma, L-2020, Lot 014K4060,(stock solution. 1 mg/mL)) in sterile PBS; final amount of Laminin perwell 20 μg. For the permissive substrate the second half of the platewas coated with 100 μl Laminin (20 μg). After an incubation of 2 hoursthe plates were washed twice with PBS and 50 μl of the pre-aggregatedcell suspension were plated in each well, supplemented with 40 μl ofmedium. Plates were incubated at 37° C. for 2 hours, and finally 10 μlof pre-diluted mAb 50 or mAb 51 solution was added to give a finalantibody concentration between 1 and 100 μg/mL. Cells were incubatedover night at 37° C., 5% CO₂, the following day fixed with 2%paraformaldehyde (SIGMA #P-6148) and stored at 4° C. for subsequentanalysis. The analysis of neurite outgrowth was performed with softwareAxioVision LE Rel. 4.1, whereby the standard evaluation parameters wereused (aggregate area and aggregate area & neurite growth area).Neurite outgrowth from NTera2 aggregates was quantified with the methoddescribed above. Results are shown in FIG. 5, significant ameliorationof neurite outgrowth can be observed at 2 μg/ml for mAb50 and at 1 μg/mlfor mAb51. Significance versus Nogo66 treatment: *=p-value<0.05;***=p-value<0.00.1Amelioration of neurite outgrowth inhibition was also obtained forantibodies Mab52, 53, 54, 55, 56, 57, 58, 59, 60, 61 and 62. Mab1 andMab4 did not ameliorate the inhibition of neurite outgrowth byAP-Nogo66.

Example 7. Deletion Mutants of the hNgR: Expression, Purification andBinding of Antibodies

hNgR was expressed as soluble protein by deletion of C-terminal aminoacids to prevent GPI-linker formation (450 last amino acid) and membraneattachment and by using a secretion signal to potentially increase theamount of secreted protein. The expression system used was based ontransient transfection of expression plasmids in 293F cells. His-taggedsecreted proteins were captured by using Ni-NTA (Nickel-nitrilotriaceticacid) beads. Eluted proteins were analyzed for purity and size by PAGEand Western Blot using a his-tagged unrelated protein (RGM A-His) as anegative control. To better adjust for NgR-protein in thesepreparations, the amount of NgR was measured by dot blots using apolyclonal antibody (AF1208) to the hNgR expected to detect all deletionmutants. Dot blots using the adjusted amount of protein for thedifferent NgR deletion mutants were performed. Dot blots were used forimmundetection with the listed antibodies. Binding of antibodies to theNgR-deletion mutants was further characterized using cell culturesupernatants of 293F-cells expressing NgR deletion mutants as a sourceof non-purified NgR

(i) Generation of NgR-deletion mutants according to Fournier et al.(Truncated Soluble Nogo Receptor Binds Nogo-66 and Blocks Inhibition ofAxon Growth by Myelin; Alyson E. Fournier, Graham C. Gould, Betty P.Liu, Stephen M. Strittmatter; The Journal of Neuroscience, Oct. 15,2002, 22(20):8876-8883). Seven (7) deletion mutants of the human NgRhave been described in the literature (Fournier et al.). The sevenmutant constructs were generated from pSecTag2A IgK/hNgR 27-450/Myc/His.This construct contains the coding region for amino acids 27-450 of thehuman NgR fused to an IgK leader peptide and a C-terminal Myc- and Histag in a pSecTag2A vector. The following constructs were generated:

pSecTag2AigK/hNgR 27-450/Myc/His

pSecTag2AigK/hNgR 58-450/Myc/His

pSecTag2AigK/hNgR27-450/Δ 58-106/Myc/His

pSecTag2AigK/hNgR27-450/Δ 106-155/Myc/His

pSecTag2AigK/hNgR27-450/Δ 155-202/Myc/His

pSecTag2AigK/hNgR27-450/Δ 203-250/Myc/His

pSecTag2AigK/hNgR27-450/Δ 260-310/Myc/His

pSecTag2AigK/hNgR 27-310/Myc/His

All constructs except pSecTag2AigK/27-310/Myc/His were generated usingthe QuikChange II XL Site Directed Mutagenesis Kit (Stratagene, #200521)and

transformations were performed in E. coli XL10 Gold cells. ForpSecTag2AigK/NgR/27-310/Myc/His the according region of the codingsequence was amplified and cloned into pSecTag2A. The followingmutagenesis and amplification primers were used for deleting thedifferent parts of the NgR coding region.

1. pSecTag2AigK/hNgR 58-450/Myc/His Mey 1008: sense primerGCCAGGCGCGCCGTACGAAGCTTATGCGCCAGCCAGCGCATCTTCCTGC ACGGC vector sequencehNgR sequence starting with amino acid A58 Mey 1009: antisense primerGCCGTGCAGGAAGATGCGCTGGCTGGCGCATAAGCTTCGTACGGCGCGC CTGGC vector sequencehNgR sequence starting with amino acid A58 2.pSecTag2AigK/hNgR27-450/Δ 58-106/Myc/His Mey 1014: sense primerGCTGTGCCCGTGGGCATCCCTGCTGCCCTCCTGGAGCAGCTGGACCTCAGCGATAATGC hNgRsequence up to amino acid A58 hNgR sequence starting from amino acidL106 Mey 1015: antisense primerGCATTATCGCTGAGGTCCAGCTGCTCCAGGAGGGCAGCAGGGATGCCCACGGGCACAGC hNgRsequence up to amino acid A58 hNgR Sequenz starting from amino acid L1063. pSecTag2AigK/hNgR27-450/Δ 106-154/Myc/His Mey 1021: sense primerGCGGCTGCCTTCACTGGCCTGGCCGCCCTGCAGTACCTCTACCTGCAGGA CAACGC hNgR sequenceup to amino acid A105 hNgR sequence starting from amino acid A155 Mey1022: antisense primerGCGTTGTCCTGCAGGTAGAGGTACTGCAGGGCGGCCAGGCCAGTGAAGGC AGCCGC hNgR sequenceup to amino acid A105 hNgR Sequence starting from amino acid A155 4.pSecTag2AigK/hNgR27-450/Δ 155-202/Myc/His Mey 1023: sense primerCGGGGCTGTTCCGCGGCCTGGCTAGCCTCGACCGTCTCCTACTGCACCAG AACCGC hNgR sequenceup to amino acid A154 hNgR sequence starting from amino acid S203 Mey1024: antisense primerGCGGTTCTGGTGCAGTAGGAGACGGTCGAGGCTAGCCAGGCCGCGGAACA GCCCCG hNgR sequenceup to amino acid A154 hNgR Sequence starting from amino acid S203 5.pSecTag2AigK/hNgR27-450/Δ 203-250/Myc/His Mey 1025: sense primerGAGCGCGCCTTCCGTGGGCTGCACGCCCTGCAGTACCTGAGGCTCAACG ACAACC hNgR sequenceup to amino acid H202 hNgR sequence starting from amino acid A251 Mey1026: antisense primer GGTTGTCGTTGAGCCTCAGGTACTGCAGGGCGTGCAGCCCACGGAAGGCGCGCTC hNgR sequence up to amino acid H202 hNgR Sequence starting fromamino acid A251 6. pSecTag2AigK/hNgR27-450/Δ 260-310/Myc/His Mey 1027:sense primer GCGTGCCCTGCAGTACCTGAGGCTCAACGACGTGGCCACCGGCCCTTACCATCCCATCTG hNgR sequence up to amino acid D259 hNgR sequence starting fromamino acid V311 Mey 1028: antisense primerCAGATGGGATGGTAAGGGCCGGTGGCCACGTCGTTGAGCCTCAGGTACTGCA hNgR sequence up toamino acid D259 hNgR Sequence starting from amino acid V311 7.pSecTag2AigK/hNgR27-310/Myc/His Mey 1016: sense primerCCCCAAGCTTATGCCCAGGTGCCTGC hNgR sequence starting from amino acid C27Mey 1030: antisense primer CCCCGAATTCCAGCGCAGCCCTGCAGGTC hNgR sequenceup to amino acid A310See a schematic representation in FIG. 6.

(ii) Expression of NgR-Deletion Mutants.

Deletion of C-terminal amino acids in the human NgR leads to loss ofmembrane anchor properties and presence of a soluble receptor protein inthe cell supernatant. At the N-terminus the signal peptide of the hNgR(amino acids 1-27) was replaced by the signal peptide encoded in thevector. Therefore a N-terminal and C-terminal deletion variant startingat amino acid 27 of the hNgR and ending at amino acid 450 of the hNgR(hNgR27-450) was used as a basis for all these mutants to enable thepresence of soluble proteins in the cell supernatants. Additionallythese mutants did contain a short amino acid tag to enable purificationof these proteins. The DNA of the hNgR mutants were transientlyexpressed in 293F cells. Cell supernatants were harvested bycentrifugation after 72 hrs. Protein purifications were done by methodsbelow. Transfections were done with the following DNA coding for the 7different mutants of the human NgR.

pSecTag2AigK/hNgR 27-450/Myc/His

pSecTag2AigK/hNgR 58-450/Myc/His

pSecTag2AigK/hNgR27-450/Δ 58-106/Myc/His

pSecTag2AigK/hNgR27-450/Δ 106-155/Myc/His

pSecTag2AigK/hNgR27-450/Δ 155-202/Myc/His

pSecTag2AigK/hNgR27-450/Δ 203-250/Myc/His

pSecTag2AigK/hNgR27-450/Δ 260-310/Myc/His

pSecTag2AigK/hNgR 27-310/Myc/His

The DNA of the hNgR mutants were transiently expressed in 293F cells.

(iii) Purification of NgR Proteins Using Ni-Chelate Affinity (Ni-NTA)

Ni-NTA superflow beads (Qiagen Qiagen #1018611) were used. Beads werewashed 3 times in PBS (phosphate buffered saline, Invitrogen) bycentrifugation of bead suspension at 13500 rpm, discarding thesupernatants, resuspending the beads in fresh PBS. 200 μl of beadsuspension were used for 30 ml cell culture supernatant. Beads wereincubated with cell culture supernatants overnight at 4° C. on arotator, 60 rpm) and were centrifuged after the incubation (10 min, 3000rpm) to pellet the beads. Supernatant was discarded and the beads washed3 times with PBS. Bound proteins were eluted from the beads using 250 μlelution buffer (PBS, 160 mM NaCl, 150 mM Imidazol). After 30 minincubation on a rotator at room temperature beads were pelleted bycentrifugation at 13.500 rpm for 3 min. Supernatant was taken. Theeluted protein was frozen at −20° C. for further analysis.The data from dot blot experiments with deletion mutants of the hNgR aresummarized in table 6.

TABLE 6 Mutants Antibodies 1 2 3 4 5 6 7 8 AF1208 +++ +++ +++ +++ ++ ++++++ ++ MAB1 +++ +++ +++ +++ +++ +++ +++ ~ MAB4 +++ +++ +++ +++ +++ ++++++ ~ MAB50 ++ − − − − +++ − ~ MAB51 ++ − − − − +++ − ~ MAB52 ++ − − − −+++ − ++ MAB53 ++ − − − − +++ − ++ MAB54 + − − − − + − ~ MAB55 + − − −− + − ~ MAB57 ~ − − − − − − − MAB58 ++ − − − ~ ++ − ~ MAB59 ++ − − − ~++ − ~ MAB60 +++ − − − ~ ++ − + MAB61 + − − − − + − + MAB62 + − − ~ + −~ +++: very strong signal; ++: strong signal; +: signal; −: no signal;~: no relevant signal.

Example 8 A. Competition of MAG-Fc Binding to NgR-Fc

To further characterize the monoclonal antibodies a competition assaysimilar to the binding assay of Example 2 was used to test the abilityof two mAbs produced in Example 1 to inhibit MAG-Fc binding to humanNgR-Fc. We immobilized NgR-Fc (0.2 μg/well, R&D Systems) on 96-wellmicrotiter plates (Nunc Maxisorb) in 50 mM Na-carbonate buffer pH 9 overnight at 4° C. followed by a 2 h blocking step with 2% BSA in Tris-HCL,pH 7.2 at room temperature. MAG-Fc (R&D Systems recombinant Rat MAG/FcChimera, Catalog #538-MG) was labeled with horseradish peroxidase by theZenon human IgG labeling kit (Molecular Probes). The wells received aconstant concentration of labeled MAG-Fc (final concentration 50 ng/mlin Tris-HCL, pH 7.2 with 0.1% BSA) and the indicated concentrations ofmAb 50 and mAb 51. This was incubated for 60 min at room temperature andusing unlabeled MAG-Fc and NgR-Fc as controls. During each incubationstep plates were washed with washing buffer (10 mM Tris-HCl, pH 7.2 and0.05% Tween 20). Labeled MAG-Fc was developed using3,3′,5,5′-tetramethylbenzidine substrate (TMB, Pierce) under standardconditions. As shown in FIG. 7, mAb 50 (closed triangles) and mAb51(open triangles) did not inhibit the binding of MAG-Fc to human NgR.MAG-Fc and NgR-Fc competed for the binding of labeled MAG-Fc (closedcircles) to human NgR-Fc (a fusion protein comprising amino acids Met 1to Ser 447 of the human NgR and a human Fc fragment (R&D Systems);closed squares). Therefore, mAb 50 and mAb 51 did not compete with MAGfor binding to NgR under these conditions (FIG. 7). None of the otherremaining antibodies compete with MAG for binding to NgR under theseconditions.

Example 8 B. Competition of OMgp Binding to NgR-Fc

To further characterize the monoclonal antibodies a competition assaysimilar to the binding assay of Example 8a was used to test the abilityof two mAbs produced in Example 1 to inhibit oMgp binding to humanNgR-Fc. NgR-Fc (0.2 μg/well, R&D Systems) was immobilized on 96-wellmicrotiter plates (Nunc Maxisorb) in 50 mM Na-carbonate buffer pH 9 overnight at 4° C. followed by a 2 h blocking step with 2% BSA in Tris-HCL,pH 7.2 at room temperature. The wells received a constant concentrationof human OMgp (R&D Systems/1673-OM; final concentration 500 ng/ml inTris-HCL, pH 7.2 with 0.1% BSA) and the indicated concentrations of mAb50 and mAb 51. This mixture was incubated for 60 min at roomtemperature. OMgp binding was detected with anti-His-antibody labeledwith horseradish peroxidase (Roche). During each incubation step plateswere washed with washing buffer (10 mM Tris-HCl, pH 7.2 and 0.05% Tween20). Labeled anti-His antibody was developed using3,3′,5,5′-tetramethylbenzidine substrate (TMB, Pierce) under standardconditions.

As shown in Table 7, mAb 50 and mAb51 partially blocked the binding ofOMgp to human NgR (30-40% range). The other antibodies, except mAb52 andmAb59, also partially blocked the binding of OMgp to human NgR, even atconcentrations up to 80 μg/ml which is more than a 100 fold molar excess(Table 7).

TABLE 7 mAb Maximal Competition (%) 1 26 4 9 50 33 51 36 52 0 53 27 5423 55 35 56 37 57 17 58 32 59 0 60 5 62 22

Example 9. Neutralization of AP-Nogo66-Induced Neurite OutgrowthInhibition by mAB50 and mAb51 in Rat DRG Cells

Dorsal root ganglions (DRGs) from rat pups of postnatal day 3-6 wereused to investigate the action of anti-NgR antibodies on neuriteoutgrowth.

For the generation of DRGs, 6-8 rat pups were decapitated with asurgical scissor. The vertebral column was dissected from the ventralside by removing ventral organs and the vertebrae. Then, the vertebralcolumn was opened longitudinally with the help of fine scissors. Thespinal cords were removed together with the adhering DRGs and weretransferred to a 10 cm petri dish containing PBS. DRGs were dissectedfree from the spinal cord and the connecting nerve fibers by using twofine forceps and were transferred to a 35 mm Petri dish containing 1 mlPBS. After collecting all DRGs, 0.5 ml of collagenase solution (4 mg/mlcollagenase Type I, Worthington # CLS-1) in PBS was added and the DRGswere incubated for 20-30 min at 37° C. 0.5 ml of trypsin solution (0.5%trypsin (SERVA #37290) in PBS) was added and the DRGs were incubated foranother 15-25 min at 37° C. The DRGs were transferred to a 15 ml tubeand 10 ml of medium (DMEM Nut Mix F12 (Gibco #31330-038), plus 5% FCS(Gibco, heat inactivated) plus 5% horse serum (Sigma, heat inactivated)plus 1% penicillin/streptomycine (Gibco #15140-122)) was added. Aftersettlement of the DRGs to the bottom of the tube, the supernatant wasremoved. DRGs were dissociated in 2 ml medium by 3-5 passages through aPasteur pipette followed by 2-3 additional passages through a Pasteurpipette with a reduced opening. After settling of cell clumps, thesupernatant containing dissociated cells was transferred to a new tube.Cells were collected by centrifugation for 5 min at 1000 rpm,resuspended in 2 ml medium, counted, and diluted to the desired celldensity in medium supplemented with Nerve Growth factor (NGF; 62.5 μg/mlfinal concentration, Roche #1014331). 4000-7000 cells were plated in avolume of 80 μl per well of a poly-lysine coated 96 well plate (e.g.Beckton Dickinson #356640), which had been additionally coated with 100μl coating solution (1 vial of laminin (1 mg/ml Sigma # L-2020) dilutedwith 50 ml of sterile water), followed by a 30 min to 3 h incubationperiod at 37° C. Cell plating was followed by the addition of increasingconcentrations of antibody in a volume of 10 μl. After incubation for 2h in a CO₂ incubator at 37° C. 10 μl of AP-Nogo66 was added. Cells weregrown for 18-30 h and fixed by the addition of 100 μl 4%paraformaldehyde solution in PBS (phosphate-buffered saline; Gibco#14190-094), followed by incubation at 4° C. for at least 12 h. As analternative to 96 well plates, 24 well plates (e.g. Falcon #353047) wereused in conjunction with poly-lysine coated coverslips (e.g. BectonDickinson #354085) and were coated by applying 500 μl coating solution.Neurite outgrowth was visualized by indirect immunofluorescence using ananti-βIII tubulin antibody (e.g. Abcam #ab14545) in conjunction with aCy3-conjugated 2nd antibody (e.g. Jackson ImmunoResearch #715-165-151).Nuclei were stained by addition of Bisbenzimide (H33258) to the 2ndantibody. Microscopic pictures were taken at 10× magnification on a BD™Pathway Bioimager (Beckton Dickinson) and neurite length was determinedusing the AttoNO (Beckton Dickinson) software. Neurite outgrowth wasnormalized to the number of DRG per picture. FIG. 8 shows theneutralization of AP-Nogo66-induced neurite outgrowth inhibition withmAb50 as an example. FIG. 9 shows the results of an experiment using theantibodies mAb 50 and mAb 51, respectively. Application of AP-Nogo66strongly reduces the length of neurite outgrowth (2nd columns) comparedto control conditions without AP-Nogo66 (1st columns). Both antibodiesneutralized the AP-Nogo66 induced inhibition of neurite outgrowth in adose-dependent fashion. Neurite length normalized to the number of DRGbecomes statistically different at 50 μg/ml (last columns) for bothantibodies compared to AP-Nogo66 application without the antibodies (2ndcolumns).

Amelioration of neurite outgrowth inhibition was also obtained forantibodies Mab52, 53, 54, 55, 56, 57, 58, 59, 60, 61 and 62. Mab1 andMab4 did not ameliorate the inhibition of neurite outgrowth byAP-Nogo66.

From these results it can be concluded that such antibodies have thepotential to stimulate neurite growth in a growth-inhibitoryenvironment.

What is claimed is:
 1. An isolated anti-Nogo-66 receptor (NgR)monoclonal antibody comprising a heavy chain variable region of SEQ IDNO:3 and a light chain variable region of SEQ ID NO:4.
 2. The isolatedanti-NgR monoclonal antibody of claim 1, wherein the antibody comprisesa kappa light chain constant region.
 3. The isolated anti-NgR monoclonalantibody of claim 1 or 2, wherein the antibody comprises a heavy chainconstant region of an IgG isotype.
 4. The isolated anti-NgR monoclonalantibody of claim 3, wherein the IgG isotype is an IgG2a isotype.
 5. Theisolated anti-NgR monoclonal antibody of claim 1, wherein the antibodyis selected from the group consisting of a mouse antibody, a humanizedantibody, a fully human, and a chimeric antibody.
 6. The isolatedanti-NgR monoclonal antibody of claim 1, wherein the antibody is human.7. A pharmaceutical composition comprising the anti-NgR monoclonalantibody of any one of claims 1 to 6 and a pharmaceutically acceptablecarrier.
 8. An isolated anti-Nogo-66 receptor (NgR) monoclonal antibodycomprising a heavy chain variable region of SEQ ID NO:5 and a lightchain variable region of SEQ ID NO:6.
 9. The isolated anti-NgRmonoclonal antibody of claim 8, wherein the antibody comprises a kappalight chain constant region.
 10. The isolated anti-NgR monoclonalantibody of claim 8 or 9, wherein the antibody comprises a heavy chainconstant region of an IgG isotype.
 11. The isolated anti-NgR monoclonalantibody of claim 10, wherein the IgG isotype is an IgG2a isotype. 12.The isolated anti-NgR monoclonal antibody of claim 8, wherein theantibody is selected from the group consisting of a mouse antibody, ahumanized antibody, a fully human, and a chimeric antibody.
 13. Theisolated anti-NgR monoclonal antibody of claim 12, wherein the antibodyis human.
 14. A pharmaceutical composition comprising the anti-NgRmonoclonal antibody of any one of claims 8 to 13 and a pharmaceuticallyacceptable carrier.